Gambogic acid, analogs and derivatives as activators of caspases and inducers of apoptosis

ABSTRACT

The present invention is directed to gambogic acid, analogs and derivatives thereof, represented by the general Formulae I-III:  
                 
 
     wherein R 1 -R 5  are defined herein. The present invention also relates to the discovery that compounds having Formula I-III are activators of caspases and inducers of apoptosis. Therefore, the activators of caspases and inducers of apoptosis of this invention can be used to induce cell death in a variety of clinical conditions in which uncontrolled cell growth and spread of abnormal cells occurs.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention is in the field of medicinal chemistry. Inparticular, the invention relates to gambogic acid, novel analogs ofgambogic acid and derivatives of gambogic acid, and the discovery thatthese compounds are activators of caspases and inducers of apoptosis.The invention also relates to the use of these compounds astherapeutically effective anti-cancer agents.

[0003] 2. Description of Background Art

[0004] Organisms eliminate unwanted cells by a process variously knownas regulated cell death, programmed cell death or apoptosis. Such celldeath occurs as a normal aspect of animal development as well as intissue homeostasis and aging (Glucksmann, A., Biol. Rev. CambridgePhilos. Soc. 26:59-86 (1951); Glucksmann, A., Archives de Biologie76:419-437 (1965); Ellis, et al., Dev. 112:591-603 (1991); Vaux, et al.,Cell 76:777-779 (1994)). Apoptosis regulates cell number, facilitatesmorphogenesis, removes harmful or otherwise abnormal cells andeliminates cells that have already performed their function.Additionally, apoptosis occurs in response to various physiologicalstresses, such as hypoxia or ischemia (PCT published applicationWO96/20721).

[0005] There are a number of morphological changes shared by cellsexperiencing regulated cell death, including plasma and nuclear membraneblebbing, cell shrinkage (condensation of nucleoplasm and cytoplasm),organelle relocalization and compaction, chromatin condensation andproduction of apoptotic bodies (membrane enclosed particles containingintracellular material) (Orrenius, S., J. Internal Medicine 237:529-536(1995)).

[0006] Apoptosis is achieved through an endogenous mechanism of cellularsuicide (Wyllie, A. H., in Cell Death in Biology and Pathology, Bowenand Lockshin, eds., Chapman and Hall (1981), pp. 9-34). A cell activatesits internally encoded suicide program as a result of either internal orexternal signals. The suicide program is executed through the activationof a carefully regulated genetic program (Wyllie, et al., Int. Rev. Cyt.68:251 (1980); Ellis, et al., Ann. Rev. Cell Bio. 7:663 (1991)).Apoptotic cells and bodies are usually recognized and cleared byneighboring cells or macrophages before lysis. Because of this clearancemechanism, inflammation is not induced despite the clearance of greatnumbers of cells (Orrenius, S., J. Internal Medicine 237:529-536(1995)).

[0007] It has been found that a group of proteases are a key element inapoptosis (see, e.g. Thornberry, Chemistry and Biology 5:R97-R103(1998); Thornberry, British Med. Bull. 53:478-490 (1996)). Geneticstudies in the nematode Caenorhabditis elegans revealed that apoptoticcell death involves at least 14 genes, two of which are thepro-apoptotic (death-promoting) ced (for cell death abnormal) genes,ced-3 and ced-4. CED-3 is homologous to interleukin 1 beta-convertingenzyme, a cysteine protease, which is now called caspase-1. When thesedata were ultimately applied to mammals, and upon further extensiveinvestigation, it was found that the mammalian apoptosis system appearsto involve a cascade of caspases, or a system that behaves like acascade of caspases. At present, the caspase family of cysteineproteases comprises 14 different members, and more may be discovered inthe future. All known caspases are synthesized as zymogens that requirecleavage at an aspartyl residue prior to forming the active enzyme.Thus, caspases are capable of activating other caspases, in the mannerof an amplifying cascade.

[0008] Apoptosis and caspases are thought to be crucial in thedevelopment of cancer (Apoptosis and Cancer Chemotherapy, Hickman andDive, eds., Humana Press (1999)). There is mounting evidence that cancercells, while containing caspases, lack parts of the molecular machinerythat activates the caspase cascade. This makes the cancer cells losetheir capacity to undergo cellular suicide and the cells becomecancerous. In the case of the apoptosis process, control points areknown to exist that represent points for intervention leading toactivation. These control points include the CED-9-BCL-like andCED-3-ICE-like gene family products. These are intrinsic proteins thatregulate the decision of a cell to survive or die and they execute partof the cell death process itself (see Schmitt, et al., Biochem. Cell.Biol. 75:301-314 (1997)), BCL-like proteins include BCL-XL andBAX-alpha, which appear to function upstream of caspase activation.BCL-XL appears to prevent activation of the apoptotic protease cascade,whereas BAX-alpha accelerates activation of the apoptotic proteasecascade.

[0009] It has been shown that chemotherapeutic (anti-cancer) drugs cantrigger cancer cells to undergo suicide by activating the dormantcaspase cascade. This may be a crucial aspect of the mode of action ofmost, if not all, known anticancer drugs (Los et al., Blood, Vol. 90, No8:3118-3129 (1997); Friesen, et al., Nat. Med. 2:574 (1996)). Themechanism of action of current antineoplastic drugs frequently involvesan attack at specific phases of the cell cycle. In brief, the cell cyclerefers to the stages through which cells normally progress during theirlifetimes. Normally, cells exist in a resting phase termed G_(o). Duringmultiplication, cells progress to a stage in which DNA synthesis occurs,termed S. Later, cell division, or mitosis occurs, in a phase called M.Antineoplastic drugs such as cytosine arabinoside, hydroxyurea,6-mercaptopurine, and methotrexate are S phase specific, whereasantineoplastic drugs such as vincristine, vinblastine, and paclitaxelare M phase specific. Many antineoplastic drugs slow growing tumors. Forexample, colon cancers exist primarily in the G_(o) phase, whereasrapidly proliferating normal tissues, for example bone marrow, existprimarily in the S or M phase. Thus, a drug like 6-mercaptopurine cancause bone marrow toxicity while remaining ineffective toward a slowgrowing tumor. Other aspects of the chemotherapy of neoplastic diseasesare known to those skilled in the art (see, e.g., Hardman, et al., eds.,Goodman and Gilman's The Pharmacological Basis of Therapeutics, NinthEdition, McGraw-Hill, N.Y. (1996), pp. 1225-1287). Thus, it is clearthat the possibility exists for the activation of the caspase cascade,although the exact mechanisms for doing so are not clear at this point.It is equally clear that insufficient activity of the caspase cascadeand consequent apoptotic events are implicated in various types ofcancer. The development of caspase cascade activators and inducers ofapoptosis is a highly desirable goal in the development oftherapeutically effective antineoplastic agents. Moreover, sinceautoimmune diseases and certain degenerative diseases also involve theproliferation of abnormal cells, therapeutic treatment for thesediseases could also involve the enhancement of the apoptotic processthrough the administration of appropriate caspase cascade activators andinducers of apoptosis.

[0010] Gambogic acid was isolated from gamboge and the structure wasdeduced from the ¹H NMR spectrum and by comparison with morellin, whichalso has the xanthone core of gambogic acid (Ahmad, S. A., et al. J.Chem. Soc. (C) 772-779 (1966); Ollis, W. D., et al. Tetrahedron,21:1453-1470 (1965)).

[0011] Asano J., et al., Phytochemistry, 41:815-820 (1996), reported theisolation of several xanthones, including gambogic acid from gamboge.They reported that gambogic acid is cytotoxic to both HeLa and HELcells.

[0012] Lin, L. -J., et al., Magn. Reson. Chem. 31:340-347 (1993),reported the isolation of gambogic acid, as well as isogambogic acid andisomorellinol. All three compounds were reported to be cytotoxic againstKB and KB-V1 cell lines.

SUMMARY OF THE INVENTION

[0013] The present invention is related to the discovery that gambogicacid, its analogs and derivatives, as represented in Formulae I-III, areactivators of the caspase cascade and inducers of apoptosis. Thereforethe first aspect of the present invention is directed to the use ofcompounds of Formulae I-III as inducers of apoptosis.

[0014] A second aspect of the present invention is to provide a methodfor treating, preventing or ameliorating neoplasia and cancer byadministering a compound of Formulae I-III to a mammal in need of suchtreatment.

[0015] A number of compounds within the scope of the present inventionare novel compounds. Therefore, a third aspect of the present inventionis to provide novel compounds of Formulae I-III, and to also provide forthe use of these novel compounds for treating, preventing orameliorating neoplasia and cancer.

[0016] A fourth aspect of the present invention is to provide apharmaceutical composition useful for treating disorders responsive tothe induction of apoptosis, containing an effective amount of a compoundof Formulae I-III in admixture with one or more pharmaceuticallyacceptable carriers or diluents.

[0017] A fifth aspect of the present invention is directed to methodsfor the isolation and preparation of novel compounds of Formulae I-III.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIGS. 1A-C depict photographs of T47D human breast cancer cellstreated with gambogic acid: control cells (FIG. 1A); cells treated with2.5 μM of gambogic acid for 2 h (FIG. 1B); cells treated with 2.5 μM ofgambogic acid for 6 h (FIG. 1C).

[0019] FIGS. 2A-B depict fluorescent photographs of T47D human breastcancer cells treated with gambogic acid and stained with a fluorescentDNA probe: control cells (FIG. 2A); cells treated with 10 μM of gambogicacid for 24 h (FIG. 2B).

[0020] FIGS. 3A-C depict photographs of Jurkat leukemia cells treatedwith gambogic acid: control cells (FIG. 3A); cells treated with 10 μM ofgambogic acid for 30 min (FIG. 3B); cells treated with 10 μM of gambogicacid in the presence of 10 μM of caspase inhibitor cbz-Val-Asp-fink(FIG. 3C).

[0021]FIG. 4 depicts the caspase activity in T47D human breast cancercells and MRC5 human non-transformed fibroblast cells treated for 2 hwith different concentrations of gambogic acid.

[0022] FIGS. 5A-D depict western blots of poly(ADP)ribose polymerase(PARP) cleavage.

[0023]FIG. 5A, Jurkat leukemia cells: (a) DMSO control, (b) treated with1 μM of staurosporine for 2 h, (c) inactive control, (d) treated with2.5 μM of gambogic acid for 2 h.

[0024]FIG. 5B, HL-60 human leukemia cancer cells: (a) DMSO control, (b)treated with 1 μM of staurosporine for 2 h, (c) inactive control, (d)treated with 2.5 μM of gambogic acid for 2 h.

[0025]FIG. 5C, T47D human breast cancer cells: (a) DMSO control, (b)treated with 1 μM of staurosporine for 2 h, (c) treated with 2.5 μM ofgambogic acid for 2 h, (d) treated with 5 μM of gambogic acid for 2 h,(e) DMSO control, (f) treated with 1 μM of staurosporine for 4 h, (g)treated with 2.5 μM of gambogic acid for 4 h, (h) treated with 5 μM ofgambogic acid for 4 h.

[0026]FIG. 5D, PC3 human prostate cancer cells: (a) DMSO control, (b)treated with 1 μM of staurosporine for 2 h, (c) treated with 2.5 μM ofgambogic acid for 2 h, (d) treated with 5 μM of gambogic acid for 2 h,(e) DMSO control, (f) treated with 1 μM of staurosporine for 4 h, (g)treated with 2.5 μM of gambogic acid for 4 h, (h) treated with 5 μM ofgambogic acid for 4 h.

DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention arises out of the discovery that gambogicacid is a potent and highly efficaceous activator of the caspase cascadeand inducer of apoptosis. Therefore gambogic acid is useful for treatingdisorders responsive to induction of apoptosis.

[0028] There are many functional groups in the structure of gambogicacid which can be modified. These include, but are not limited to, thecarboxyl group, which can be converted to an ester, amide, ketone oralcohol and other functional groups; the ester and amide, in turn, mayalso contain other functional groups, such as the carboxyl of an aminoacid, which can be further modified; the hydroxy group, may be convertedto an ether, ester or other functional groups; the carbon-carbon doublebond between C-9 and C-10 is part of an α,β-unsaturated ketone, whichcan react with a nucleophile, be reduced to a carbon-carbon single bond,or may be converted to an epoxide, which in turn may undergo furtherreaction; the carbon-carbon double bond between C-27 and C-28 is part ofan α,β-unsaturated carboxyl, that may also react with a nucleophile, bereduced to a carbon-carbon single bond, or may be converted to acyclopropane ring, which in turn may undergo further reaction; the twoisoprene carbon-carbon double bonds at C-37/C-38 and C-32/C-33, may alsobe reduced to a carbon-carbon single bond, be cleaved to form analdehyde group or a carboxyl group, both of which may be modified toother functional groups, or be converted to an epoxide, which in turnmay undergo further reaction; the carbon-carbon double bond between C-3and C-4 may also be reduced to a carbon-carbon single bond, or beconverted to an epoxide that may undergo further reaction; the ketonegroup at C-12 may be reduced to an alcohol, or may be converted to anoxime, a semicarbazone, or an amino group; the other ketone group mayalso be reduced, or may be converted to other functional groups. Inshort, many derivatives of gambogic acid can be prepared.

[0029] In addition, analogs of gambogic acid, including isomorellin,morellic acid, desoxymorellin, gambogin, morelline dimethyl acetal,isomoreollin B Moreollic acid, gambogenic acid, gambogenin,isogambogenin, desoxygambogenin, gambogenin dimethyl acetal, gambogellicacid, hanburin (Asano, J., et al., Phytochemistry 41:815-820 (1996)),isogambogic acid, isomorellinol (Lin, L. -J., et al., Magn. Reson. Chem.31:340-347 (1993)) and neo-gambogic acid (Lu, G. B., et al., Yao HsuehHsueh Pao 19:636-639 (1984)) can be isolated from gamboge. Other analogsof gambogic acid, including morellin, desoxymorellin, dihydroisomorellin(Bhat et al. Indian J. Chem. 2:405-409 (1964)) and moreollin (Rao et al.Proc. Indian Acad. Sci. 87A:75-86 (1978)), can be isolated from the seedof Garcinia morella. Morellinol can be isolated from the bark ofGarcinia morella (Adawadkar et al. Indian J Chem. 14B:19-21 (1976)).Gaudichaudiones (A-H) and gaudichaudiic acids A-E can be isolated fromthe leaves of Garcinia gaudichaudii (Guttiferae) (Cao, S. -G., et al.,Tetrahedron 54(36):10915-10924 (1998) and Cao, S. -G., et al.,Tetrahedron Lett. 39(20):3353-3356 (1998)), and forbesione can beisolated from Garcinia forbesii (Leong, Y. -W., et al., J. Chem. Res.,Synop. 392-393 (1996)).

[0030] The present invention, therefore, also arises out of thediscovery that novel derivatives and analogs of gambogic acid are alsoactivators of the caspase cascade and inducers of apoptosis. Thereforethese derivatives and analogs of gambogic acid are useful for treatingdisorders responsive to the induction of apoptosis.

[0031] Specifically, compounds useful in this aspect of the presentinvention are gambogic acid, its analogs and derivatives as representedby Formulae I-III:

[0032] or pharmaceutically acceptable salts or prodrugs thereof,wherein:

[0033] the dotted lines are single bonds, double bonds or an epoxygroup;

[0034] X together with the attached carbon is a methylene, carbonyl,hydroxymethinyl, alkoxymethinyl, aminomethinyl, an oxime, a hydrazone,an arylhydrazone or semicarbazone;

[0035] Y together with the attached carbon is a methylene, carbonyl,hydroxymethinyl, alkoxymethinyl, aminomethinyl, an oxime, a hydrazone,an arylhydrazone or semicarbazone;

[0036] R₁ is formyl, methylenehydroxy, carboxy, acyl (R_(a)CO),optionally substituted alkoxycarbonyl (R_(a)OCO), optionally substitutedalkylthiocarbonyl, optionally substituted aminocarbonyl (carbamyl,R_(b)R_(c)CO) or hydroxyaminocarbonyl, where R_(a) is hydrogen,optionally substituted lower alkyl, optionally substituted aryl,optionally substituted lower aralkyl group or N-succinimidyl; R_(b) andR_(c) are independently hydrogen, optionally substituted heteroalkyl,optionally substituted lower alkyl, optionally substituted aryl,optionally substituted heteroaryl or optionally substituted loweraralkyl groups; or R_(b) and R_(c) may be taken together with theattached N to form an optionally substituted, saturated or partiallysaturated 5-7 membered heterocyclo group, including piperidine,morpholine and piperazine.

[0037] R₂ is hydrogen, optionally substituted alkyl, acyl (R_(a)CO),carbamyl (R_(b)R_(c)NCO) or sulfonyl (R_(d)SO₂), where R_(a), R_(b) andR_(c) are defined above; R_(d) is hydrogen, optionally substituted loweralkyl, optionally substituted aryl, or optionally substituted loweraralkyl groups;

[0038] R₃ is hydrogen or prenyl;

[0039] R₄ is hydrogen, halogen, hydroxy, optionally substituted alkyl,cycloalkyl, alkoxy, alkylthio or amino;

[0040] R₅ is hydrogen, optionally substituted alkyl or acyl (R_(a)CO),carbamyl (R_(b)R_(c)NCO) or sulfonyl (R_(d)SO₂), where R_(a), R_(b),R_(c) and R_(d) are defined above.

[0041] Preferred compounds falling within the scope of Formula I includecompounds wherein R₁ is formyl, acetyl, propionyl, carboxy,methoxy-carbonyl, ethoxycarbonyl, methylthiocarbonyl, ethylthiocarbonyl,butylthiocarbonyl, dimethylcarbamyl, diethylcarbamoyl,1-piperidinylcarbonyl, N-methyl-N′-piperazinylcarbonyl,2-(dimethylamino)-ethylcarbamyl or N-morpholinylcarbonyl; R₂ ishydrogen, formyl, acetyl, dimethylcarbamyl, diethylcarbamyl,2-(dimethylamino)ethylcarbamyl, 1-piperidinylcarbonyl,N-methyl-N′-piperazinylcarbonyl, N-morpholinyl-carbonyl, methylsulfonyl,ethylsulfonyl, phenylsulfonyl, methyl, ethyl, 2-piperidinylethyl,2-morpholinylethyl, 2-(dimethylamino)ethyl, or 2-(diethylamino)ethyl; Xand Y is O; R₃ is prenyl; and the dotted lines are double bonds or anepoxy group. If the double bond is present at C27-28, it is preferredthat it has the Z configuration.

[0042] Preferred compounds falling within the scope of Formula IIinclude compounds wherein R₁ is formyl, acetyl, propionyl, carboxy,methoxy-carbonyl, ethoxycarbonyl, methylthiocarbonyl, ethylthiocarbonyl,butylthiocarbonyl, dimethylcarbamyl, diethylcarbamyl,N-piperidinylcarbonyl, N-methyl-N′-piperazinylcarbonyl,2-(dimethylamino)ethylcarboxy or N-morpholinylcarbonyl; R₂ is hydrogen,formyl, acetyl, dimethylcarbamyl, diethylcarbamyl,2-(dimethylamino)ethylcarbamyl, 1-piperidinylcarbonyl,N-methyl-N′-piperazinylcarbonyl, N-morpholinylcarbonyl, methylsulfonyl,ethylsulfonyl, phenylsulfonyl, methyl, ethyl, 2-piperidinylethyl,2-morpholinylethyl, 2-(dimethylamino)ethyl, or 2-(diethylamino)ethyl;and R₄ is methyl, ethyl, phenyl, chloro, bromo, hydroxy, hydrogen,methoxy, ethoxy, methylthio, ethylthio, butylthio, dimethylamino,diethylamino, piperidinyl, pyrrolidinyl, imidazolyl, pyrazolyl,N-methylpiperazinyl, 2-(dimethylamino)ethylamino or morpholinyl; X and Yis O; R₃ is prenyl; and the dotted lines are double bonds. If the doublebond is present at C27-28, it is preferred that it has the Zconfiguration.

[0043] Preferred compounds falling within the scope of Formula IIIinclude compounds wherein R₁ is formyl, acetyl, propionyl, carboxy,methoxy-carbonyl, ethoxycarbonyl, methylthiocarbonyl, ethylthiocarbonyl,butylthiocarbonyl, dimethylcarbamyl, diethylcarbamyl,N-piperidinylcarbonyl, N-methyl-N′-piperazinylcarbonyl,2-(dimethylamino)ethylcarbamyl or N-morpholinylcarbonyl; R₂ is hydrogen,formyl, acetyl, dimethylcarbamyl, diethylcarbamyl,2-(dimethylamino)ethylcarbamyl, 1-piperidinylcarbonyl,N-methyl-N′-piperazinylcarbonyl, N-morpholinylcarbonyl, methylsulfonyl,ethylsulfonyl, phenylsulfonyl, methyl, ethyl, 2-piperidinylethyl,2-morpholinylethyl, 2-(dimethylamino)ethyl, or 2-(diethylamino)ethyl; R₅is hydrogen, formyl, acetyl, dimethylcarbamyl, diethylcarbamyl,2-(dimethylamino)ethylcarbamyl, 1-piperidinylcarbonyl,N-methyl-N′-piperazinylcarbonyl, N-morpholinylcarbonyl, methylsulfonyl,ethylsulfonyl, phenylsulfonyl, methyl, ethyl, 2-piperidinylethyl,2-morpholinylethyl, 2-(dimethylamino)ethyl, or 2-(diethylamino)ethyl; Xand Y is O; R₃ is prenyl; and the dotted lines are double bonds. If thedouble bond is present at C27-28, it is preferred that it has the Zconfiguration.

[0044] Exemplary preferred compounds that may be employed in the methodof invention include, without limitation:

[0045] Gambogic acid;

[0046] Methyl gambogate;

[0047] 9,10-Dihydrogambogic acid;

[0048] 9,10-Dihydrogambogyl (4-methylpiperazine);

[0049] 9,10-Dihydrogambogyl (2-dimethylaminoethylamine);

[0050] Gambogyl diethylamine;

[0051] Gambogyl dimethylamine;

[0052] Gambogyl amine;

[0053] Gambogyl hydroxyamine;

[0054] Gambogyl piperidine;

[0055] 6-Methoxy-gambogic acid;

[0056] 6-(2-Dimethylaminoethoxy)-gambogic acid;

[0057] 6-(2-Piperidinylethoxy)-gambogic acid;

[0058] 6-(2-Morpholinylethoxy)-gambogic acid;

[0059] 6-Methoxy-gambogyl piperidine;

[0060] Gambogyl morpholine;

[0061] Gambogyl (2-dimethylaminoethylamine);

[0062] 10-Morpholinyl-gambogyl morpholine;

[0063] 10-Morpholinyl-gambogyl piperidine;

[0064] 10-(4-Methylpiperazinyl)-gambogyl piperidine;

[0065] 10-(4-Methylpiperazinyl)-gambogyl morpholine;

[0066] 10-Piperidinyl-gambogyl piperidine;

[0067] 10-(4-Methylpiperazinyl)-gambogyl (4-methylpiperazine);

[0068] Gambogyl (4-methylpiperazine);

[0069] Methyl-6-methoxy-gambogate;

[0070] Gambogenic acid;

[0071] Gambogenin;

[0072] 10-Methoxy-gambogic acid;

[0073] 10-Butylthio-gambogic acid;

[0074] 10-(4-Methylpiperazinyl)-gambogic acid;

[0075] 10-Pyrrolidinyl-gambogic acid;

[0076] Methyl-10-Morpholinyl-gambogate;

[0077] 10-Piperidinyl-gambogic acid;

[0078] 10-Morpholinyl-gambogic acid;

[0079] N-(2-Gambogylamidoethyl)biotinamide;

[0080] Gambogyl (2-morpholinylethylamine);

[0081] 9,10-Epoxygambogic acid;

[0082] Gambogyl (4-(2-pyridyl)piperazine);

[0083] 10-(4-(2-Pyridyl)piperazinyl)gambogyl (4-(2-pyridyl)piperazine);

[0084] 6-Acetylgambogic acid;

[0085] 10-(4-(2-Pyridyl)piperazinyl)gambogic acid;

[0086] N-Hydroxysuccinimidyl gambogate;

[0087] 8-(Gambogylamido)octanoic acid;

[0088] 6-(Gambogylamido)hexanoic acid;

[0089] 12-(Gambogylamido)dodecanoic acid;

[0090] N-Hydroxysuccinimidyl-8-(gambogylamido)octanoate;

[0091] N-Hydroxysuccinimidyl-6-(gambogylamido)hexanoate;

[0092] N-Hydroxysuccinimidyl-12-(gambogylamido)dodecanoate;

[0093] 10-Methoxy-gambogyl piperidine;

[0094] Gambogyl (4-(2-pyrimidyl)piperazine);

[0095] Gambogyl (bis(2-pyridylmethyl)amine);

[0096] Gambogyl (N-(3-pyridyl)-N-(2-hydroxybenzyl)amine);

[0097] Gambogyl (4-benzylpiperazine);

[0098] Gambogyl (4-(3,4-methylenedioxybenzyl)piperazine);

[0099] Gambogyl (N-methyl-5-(methylamino)-3-oxapentylamine);

[0100] Gambogyl (N-methyl-8-(methylamino)-3,6-dioxaoctylamine);

[0101] Gambogyl (N-ethyl-2-(ethylamino)ethylamine);

[0102] Gambogyl (4-isopropylpiperazine);

[0103] Gambogyl (4-cyclopentylpiperazine);

[0104] Gambogyl (N-(2-oxo-2-ethoxyethyl)-(2-pyridyl)methylamine);

[0105] Gambogyl (2,5-dimethylpiperazine);

[0106] Gambogyl (3,5-dimethylpiperazine);

[0107] Gambogyl (4-(4-acetylphenyl)piperazine);

[0108] Gambogyl (4-ethoxycarbonylpiperazine);

[0109] Gambogyl (4-(2-oxo-2-pyrrolidylethyl)piperazine);

[0110] Gambogyl (4-(2-hydroxyethyl)piperazine);

[0111] Gambogyl (N-methyl-2-(methylamino)ethylamine);

[0112] Gambogyl (N-methyl-2-(benzylamino)ethylamine);

[0113] Gambogyl (N-methyl-(6-methyl-2-pyridyl)methylamine);

[0114] Gambogyl (N-ethyl-2-(2-pyridyl)ethylamine);

[0115] Gambogyl (N-methyl-(2-pyridyl)methylamine);

[0116] Gambogyl (N-methyl-4-(3-pyridyl)butylamine);

[0117] Gambogyl (bis(3-pyridylmethyl)amine);

[0118] Gambogyl (2,4-dimethyl-2-imidazoline);

[0119] Gambogyl (4-methyl-homopiperazine);

[0120] Gambogyl (4-(5-hydroxy-3-oxapentyl)piperazine);

[0121] Gambogyl (3-dimethylaminopyrrolidine);

[0122] Gambogyl ((2-furanyl)methylamine);

[0123] Gambogyl (2-hydroxy-1-methyl-2-phenylethylamine);

[0124] Gambogyl (3,4,5-trimethoxybenzylamine);

[0125] Gambogyl (2-(2-methoxyphenyl)ethylamine);

[0126] Gambogyl (2-methoxybenzylamine);

[0127] Gambogyl (3,4-methylenedioxybenzylamine);

[0128] Gambogyl (2-(2,5-dimethoxyphenyl)ethylamine);

[0129] Gambogyl (2-(3-methoxyphenyl)ethylamine);

[0130] Gambogyl (3-(piperidinyl)propylamine);

[0131] Gambogyl (2-(piperidinyl)ethylamine);

[0132] Gambogyl (3,4-dimethoxybenzylamine);

[0133] Gambogyl ((2-tetrahydrofuranyl)methylamine);

[0134] Gambogyl ((N-ethyl-2-pyrrolidinyl)methylamine);

[0135] Gambogyl (2-diethylaminoethylamine);

[0136] Gambogyl (2,2-dimethyl-3-dimethylaminopropylamine);

[0137] Gambogyl ((N-ethoxycarbonyl-4-piperidinyl)amine);

[0138] Gambogyl (2-carbamylpyrrolidine);

[0139] Gambogyl (3-(homopiperidinyl)propylamine);

[0140] Gambogyl ((N-benzyl-4-piperidinyl)amine);

[0141] Gambogyl (2-(4-methoxyphenyl)ethylamine);

[0142] Gambogyl (4-oxa-hex-5-enylamine);

[0143] Gambogyl (6-hydroxyhexylamine);

[0144] Gambogyl (2-(3,5-dimethoxyphenyl)ethylamine);

[0145] Gambogyl (3,5-dimethoxybenzylamine); and

[0146] Gambogyl (2-carbamyl-2-(4-hydroxyphenyl)ethylamine).

[0147] The positions in gambogic acid are numbered according to Asano,J., et al., Phytochemistry 41:815-820 (1996), and Lin, L. -J., et al.,Magn. Reson. Chem. 31:340-347 (1993).

[0148] The present invention is also directed to novel compounds withinthe scope of Formulae I-III. These compounds include compounds ofFormula I wherein if R₁ is carboxy or methoxycarbonyl and X and Y are O,then R₂ is not hydrogen or methyl. These compounds also includecompounds of Formula II wherein if R₁ is formyl or carboxy, R₂ ishydrogen, R₃ is hydrogen and X and Y are O, then R₄ is not methoxy orethoxy. These compounds also include compounds of Formula III wherein ifR₁ is formyl or carboxy and X and Y are O, then at least one of R₂ or R₅are not hydrogen.

[0149] Exemplary preferred compounds that may be employed in thisinvention include, without limitation:

[0150] 9,10-Dihydrogambogyl (4-methylpiperazine);

[0151] 9,10-Dihydrogambogyl (2-(dimethylamino)ethylamine);

[0152] 9,10-Dihydro-12-hydroxygambogic acid;

[0153] Gambogyl diethylamine;

[0154] Gambogyl dimethylamine;

[0155] Gambogyl amine;

[0156] Gambogyl hydroxyamine;

[0157] Gambogyl piperidine;

[0158] 6-Methoxy-gambogic acid;

[0159] 6-(2-Dimethylaminoethoxy)-gambogic acid;

[0160] 6-(2-Piperidinylethoxy)-gambogic acid;

[0161] 6-(2-Morpholinylethoxy)-gambogic acid;

[0162] 6-Methoxy-gambogyl piperidine;

[0163] Gambogyl 4-morpholine;

[0164] Gambogyl 2-(dimethylamino)ethylamine;

[0165] 10-Morpholinyl-gambogyl morpholine;

[0166] 10-Morpholinyl-gambogyl piperidine;

[0167] 10-(4-Methylpiperazinyl)-gambogyl piperidine;

[0168] 10-(4-Methylpiperazinyl)-gambogyl morpholine;

[0169] 10-Piperidinyl-gambogyl piperidine;

[0170] 10-(4-Methylpiperazinyl)-gambogyl (4-methylpiperazine);

[0171] Gambogyl (4-methylpiperazine);

[0172] 10-Methoxy-gambogic acid;

[0173] 10-Butylthio-gambogic acid;

[0174] 10-(4-Methylpiperazinyl)-gambogic acid;

[0175] 10-Pyrrolidinyl-gambogic acid;

[0176] Methyl-10-Morpholinyl-gambogate;

[0177] 10-Piperidinyl-gambogic acid;

[0178] 10-Morpholinyl-gambogic acid;

[0179] 10-Cyclohexyl-gambogic acid;

[0180] 10-Methyl-gambogic acid;

[0181] N-(2-Gambogylamido-ethyl)biotinamide;

[0182] Gambogyl (2-(4-morpholinyl)ethylamine);

[0183] 9,10-Epoxygambogic acid;

[0184] Gambogyl (4-(2-pyridyl)piperazine);

[0185] 10-(4-(2-Pyridyl)piperazinyl)gambogyl(4-(2-pyridyl)piperazine);

[0186] 6-Acetylgambogic acid;

[0187] 10-(4-(2-Pyridyl)piperazinyl)gambogic acid;

[0188] N-Hydroxysuccinimidyl gambogate;

[0189] 8-(Gambogylamido)octanoic acid;

[0190] 6-(Gambogylamido)hexanoic acid;

[0191] 12-(Gambogylamido)dodecanoic acid;

[0192] N-Hydroxysuccinimidyl-8-(gambogylamido)octanoate;

[0193] N-Hydroxysuccinimidyl-6-(gambogylamido)hexanoate;

[0194] N-Hydroxysuccinimidyl-12-(gambogylamido)dodecanoate;

[0195] 10-Methoxy-gambogyl piperidine;

[0196] Gambogyl (4-(2-pyrimidyl)piperazine);

[0197] Gambogyl (bis(2-pyridylmethyl)amine);

[0198] Gambogyl (N-(3-pyridyl)-N-(2-hydroxybenzyl)amine);

[0199] Gambogyl (4-benzylpiperazine);

[0200] Gambogyl (4-(3,4-methylenedioxybenzyl)piperazine);

[0201] Gambogyl (N-methyl-5-(methylamino)-3-oxapentylamine);

[0202] Gambogyl (N-methyl-8-(methylamino)-3,6-dioxaoctylamine);

[0203] Gambogyl (N-ethyl-2-(ethylamino)ethylamine);

[0204] Gambogyl (4-isopropylpiperazine);

[0205] Gambogyl (4-cyclopentylpiperazine);

[0206] Gambogyl (N-(2-oxo-2-ethoxyethyl)-(2-pyridyl)methylamine);

[0207] Gambogyl (2,5-dimethylpiperazine);

[0208] Gambogyl (3,5-dimethylpiperazine);

[0209] Gambogyl (4-(4-acetylphenyl)piperazine);

[0210] Gambogyl (4-ethoxycarbonylpiperazine);

[0211] Gambogyl (4-(2-oxo-2-pyrrolidylethyl)piperazine);

[0212] Gambogyl (4-(2-hydroxyethyl)piperazine);

[0213] Gambogyl (N-methyl-2-(methylamino)ethylamine);

[0214] Gambogyl (N-methyl-2-(benzylamino)ethylamine);

[0215] Gambogyl (N-methyl-(6-methyl-2-pyridyl)methylamine);

[0216] Gambogyl (N-ethyl-2-(2-pyridyl)ethylamine);

[0217] Gambogyl (N-methyl-(2-pyridyl)methylamine);

[0218] Gambogyl (N-methyl-4-(3-pyridyl)butylamine);

[0219] Gambogyl (bis(3-pyridylmethyl)amine);

[0220] Gambogyl (2,4-dimethyl-2-imidazoline);

[0221] Gambogyl (4-methyl-homopiperazine);

[0222] Gambogyl (4-(5-hydroxy-3-oxapentyl)piperazine);

[0223] Gambogyl (3-dimethylaminopyrrolidine);

[0224] Gambogyl ((2-furanyl)methylamine);

[0225] Gambogyl (2-hydroxy-1-methyl-2-phenylethylamine);

[0226] Gambogyl (3,4,5-trimethoxybenzylamine);

[0227] Gambogyl (2-(2-methoxyphenyl)ethylamine);

[0228] Gambogyl (2-methoxybenzylamine);

[0229] Gambogyl (3,4-methylenedioxybenzylamine);

[0230] Gambogyl (2-(2,5-dimethoxyphenyl)ethylamine);

[0231] Gambogyl (2-(3-methoxyphenyl)ethylamine);

[0232] Gambogyl (3-(piperidinyl)propylamine);

[0233] Gambogyl (2-(piperidinyl)ethylamine);

[0234] Gambogyl (3,4-dimethoxybenzylamine);

[0235] Gambogyl ((2-tetrahydrofuranyl)methylamine);

[0236] Gambogyl ((N-ethyl-2-pyrrolidinyl)methylamine);

[0237] Gambogyl (2-diethylaminoethylamine);

[0238] Gambogyl (2,2-dimethyl-3-dimethylaminopropylamine);

[0239] Gambogyl ((N-ethoxycarbonyl-4-piperidinyl)amine);

[0240] Gambogyl (2-carbamylpyrrolidine);

[0241] Gambogyl (3-(homopiperidinyl)propylamine);

[0242] Gambogyl ((N-benzyl-4-piperidinyl)amine);

[0243] Gambogyl (2-(4-methoxyphenyl)ethylamine);

[0244] Gambogyl (4-oxa-hex-5-enylamine);

[0245] Gambogyl (6-hydroxyhexylamine);

[0246] Gambogyl (2-(3,5-dimethoxyphenyl)ethylamine);

[0247] Gambogyl (3,5-dimethoxybenzylamine); and

[0248] Gambogyl (2-carbamyl-2-(4-hydroxyphenyl)ethylamine).

[0249] Useful alkyl groups include straight-chained and branched C₁₋₁₀alkyl groups, more preferably C₁₋₆ alkyl groups. Typical C₁₋₁₀ alkylgroups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl,tert-butyl, 3-pentyl, hexyl and octyl groups, which may be optionallysubstituted.

[0250] Useful alkoxy groups include oxygen substituted by one of theC₁₋₁₀ alkyl groups mentioned above, which may be optionally substituted.

[0251] Useful alkylthio groups include sulphur substituted by one of theC₁₋₁₀ alkyl groups mentioned above, which may be optionally substituted.Also included are the sulfoxides and sulfones of such alkylthio groups.

[0252] Useful amino groups include —NH₂, —NHR₁₁, and —NR₁R₁₂, whereinR₁₁ and R₁₂ are C₁₋₁₀ alkyl or cycloalkyl groups, or R₁₁ and R₁₂ arecombined with the N to form a ring structure, such as a piperidine, orR₁₁ and R₁₂ are combined with the N and another heteroatom to form anoptionally substituted, saturated or partially saturated 5-7 memberedheterocyclo group, such as a piperazine. The alkyl group may beoptionally substituted.

[0253] Useful heteroatoms include N, O or S.

[0254] Optional substituents on the alkyl groups include one or morehalo, hydroxy, carboxyl, alkoxycarbonyl, amino, nitro, cyano, C₁-C₆acylamino, C₁-C₆ aminoacyl, C₁-C₆ acyloxy, C₁-C₆ alkoxy, aryloxy,alkylthio, C₆-C₁₀ aryl, C₄-C₇ cycloalkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₆-C₁₀ aryl(C₂-C₆)alkenyl, C₆-C₁₀ aryl(C₂-C₆)alkynyl, saturated orpartially saturated 5-7 membered heterocyclo group, or heteroaryl.

[0255] Optional substituents on the aryl, aralkyl and heteroaryl groupsinclude one or more acyl, alkylenedioxy (—OCH₂O—), halo, C₁-C₆haloalkyl, C₆-C₁₀ aryl, C₄-C₇ cycloalkyl, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₆-C₁₀ aryl(C₁-C₆)alkyl, C₆-C₁₀ aryl(C₂-C₆)alkenyl,C₆-C₁₀ aryl(C₂-C₆)alkynyl, C₁-C₆ hydroxyalkyl, nitro, amino, ureido,cyano, C₁-C₆ acylamino, hydroxy, thiol, C₁-C₆ acyloxy, azido, C₁-C₆alkoxy, or carboxy.

[0256] Useful heteroalkyl groups contain 1-10 carbon atoms and 1, 2 or 3heteroatoms. Examples of heteroalkyl groups include —CH₂CH₂O CH₂CH₃,—CH₂CH₂OCH₂CH₂OCH₂CH₃, —CH₂CH₂NHCH₃, —CH₂CH₂N(CH₂CH₃)₂,—CH₂CH₂OCH₂CH₂NHCH₃, —CH₂CH₂OCH₂CH₂OCH₂CH₂NHCH₃, —CH₂CH₂NHCH₂CH₃,—CH₂C(CH₃)₂CH₂N(CH₃)₂ or —CH₂(N-ethylpyrrolidine), which may beoptionally substituted.

[0257] Optional substituents on heteroalkyl groups include one or morehalo, hydroxy, carboxyl, amino, nitro, cyano, alkyl, C₁-C₆ acylamino,C₁-C₆ aminoacyl, C₁-C₆ acyloxy, C₁-C₆ alkoxy, aryloxy, alkylthio, C₆-C₁₀aryl, C₄-C₇ cycloalkyl, C₂-C₆ alkenyl, alkenoxy, C₂-C₆ alkynyl, C₆-C₁₀aryl(C₂-C₆)alkenyl, C₆-C₁₀ aryl(C₂-C₆)alkynyl, saturated and unsaturatedheterocyclic, or heteroaryl.

[0258] Useful aryl groups are C₆₋₁₄ aryl, especially C₆₋₁₀ aryl. TypicalC₆₋₁₄ aryl groups include phenyl, naphthyl, phenanthrenyl, anthracenyl,indenyl, azulenyl, biphenyl, biphenylenyl and fluorenyl groups.

[0259] Useful cycloalkyl groups are C₃₋₈ cycloalkyl. Typical cycloalkylgroups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl andcycloheptyl.

[0260] Useful saturated or partially saturated carbocyclic groups arecycloalkyl groups as defined above, as well as cycloalkenyl groups, suchas cyclopentenyl, cycloheptenyl and cyclooctenyl.

[0261] Useful halo or halogen groups include fluorine, chlorine, bromineand iodine.

[0262] Useful aralkyl groups include any of the above-mentioned C₁₋₁₀alkyl groups substituted by any of the above-mentioned C₆₋₁₄ arylgroups. Useful values include benzyl, phenethyl and naphthylmethyl.

[0263] Useful haloalkyl groups include C₁₋₁₀ alkyl groups substituted byone or more fluorine, chlorine, bromine or iodine atoms, e.g.fluoromethyl, difluoromethyl, trifluoromethyl, pentafluoroethyl,1,1-difluoroethyl, chloromethyl, chlorofluoromethyl and trichloromethylgroups.

[0264] Useful acylamino groups are any C₁₋₆ acyl (alkanoyl) attached toan amino nitrogen, e.g. acetamido, propionamido, butanoylamido,pentanoylamido, hexanoylamido as well as aryl-substituted C₂₋₆substituted acyl groups.

[0265] Useful acyloxy groups are any C₁₋₆ acyl (alkanoyl) attached to anoxy (—O—) group, e.g. formyloxy, acetoxy, propionoyloxy, butanoyloxy,pentanoyloxy, hexanoyloxy and the like.

[0266] Useful saturated or partially saturated 5-7 membered heterocyclogroups include tetrahydrofuranyl, pyranyl, piperidinyl, piperazinyl,pyrrolidinyl, imidazolidinyl, imidazolinyl, indolinyl, isoindolinyl,quinuclidinyl, morpholinyl, isochromanyl, chromanyl, pyrazolidinylpyrazolinyl, tetronoyl and tetramoyl groups.

[0267] Optional substitutents on the 5-7 membered heterocyclo groupsinclude one or more heteroaryl, heterocyclo, alkyl, aralkyl, cycloalkyl,alkoxycarbonyl, carbamyl, aryl or C₁-C₆ aminoacyl.

[0268] Useful heteroaryl groups include any one of the following:thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furanyl,pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxanthiinyl,2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl,pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl,indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalzinyl,naphthyridinyl, quinozalinyl, cinnolinyl, pteridinyl, carbazolyl,β-carbolinyl, phenanthridinyl, acrindinyl, perimidinyl, phenanthrolinyl,phenazinyl, isothiazolyl, phenothiazinyl, isoxazolyl, furazanyl,phenoxazinyl, 1,4-dihydroquinoxaline-2,3-dione, 7-aminoisocoumarin,pyrido[1,2-a]pyrimidin-4-one, 1,2-benzoisoxazol-3-yl, benzimidazolyl,2-oxindolyl and 2-oxobenzimidazolyl. Where the heteroaryl group containsa nitrogen atom in a ring, such nitrogen atom may be in the form of anN-oxide, e.g. a pyridyl N-oxide, pyrazinyl N-oxide, pyrimidinyl N-oxideand the like.

[0269] Optional substituents on the heteroaryl groups include one ormore heteroaryl, heterocyclo, alkyl, aralkyl, cycloalkyl,alkoxycarbonyl, carbamyl, aryl and C₁-C₆ aminoacyl.

[0270] Certain compounds of the present invention may exist asstereoisomers including optical isomers. The invention includes allstereoisomers and both the racemic mixtures of such stereoisomers aswell as the individual enantiomers that may be separated according tomethods that are well known to those of ordinary skill in the art.

[0271] Examples of pharmaceutically acceptable addition salts includeinorganic and organic acid addition salts such as hydrochloride,hydrobromide, phosphate, sulphate, citrate, lactate, tartrate, maleate,fumarate, mandelate and oxalate; and inorganic and organic base additionsalts with bases such as sodium hydroxy, Tris(hydroxymethyl)aminomethane(TRIS, tromethane) and N-methyl-glucamine.

[0272] Examples of prodrugs of the compounds of the invention includethe simple esters of carboxylic acid containing compounds (e.g. thoseobtained by condensation with a C₁₋₄ alcohol according to methods knownin the art); esters of hydroxy containing compounds (e.g. those obtainedby condensation with a C₁₋₄ carboxylic acid, C₃₋₆ dioic acid oranhydride thereof, such as succinic and fumaric anhydrides according tomethods known in the art); imines of amino containing compounds (e.g.those obtained by condensation with a C₁₋₄ aldehyde or ketone accordingto methods known in the art); and acetals and ketals of alcoholcontaining compounds (e.g. those obtained by condensation withchloromethyl methyl ether or chloromethyl ethyl ether according tomethods known in the art).

[0273] The compounds of this invention may be prepared and purifiedusing methods known to those skilled in the art, or the novel methods ofthis invention. Specifically, gambogic acid can be purified by 1)preparation of the pyridine salt of the crude extract from gamboge(resin from Garcinia hanburyi Hook) followed by repeatedrecrystallization of the salt in ethanol or 2) converting the salt tothe free acid. Using this procedure, about 10% by weight of gambogicacid with purity >99% (HPLC) can be obtained from the crude extract.Gambogic acid and analogs of gambogic acid with Formula I-III also canbe separated and purified from gamboge by repeated chromatography (SiO₂,hexane-EtOAc gradient) using a Combi Flash SG 100 separation system.

[0274] Derivatives of gambogic acid with Formula I can be prepared asillustrated by exemplary reactions in Schemes 1 and 2. Reaction ofgambogic acid with methanol in the presence of DMAP and EDC produced themethyl ester of gambogic acid (Scheme 1). Reaction of gambogic acid withpiperidine in the presence of DMAP and EDC produced the piperidinylamide of gambogic acid (Scheme 2).

[0275] Derivatives of gambogic acid with Formula I can also be preparedas illustrated by exemplary reactions in Schemes 3-5. Reaction of methylgambogate with methyl iodide in the presence of a base, such as K₂CO₃,produced the methyl-6-methoxy-gambogate (Scheme 3). Reaction of gambogicacid with acetic anhydride in pyridine produced 6-acetyl gambogic acid(Scheme 4). Reaction of gambogic acid with H₂O₂ under basic conditionsproduced 9,10-epoxygambogic acid (Scheme 5).

[0276] Derivatives of gambogic acid with Formula II can be prepared asillustrated by exemplary reactions in Schemes 6-10. Reaction of gambogylpiperidine with sodium methoxide produced the methoxy addition productof the amide (Scheme 6). Similarly, reaction of gambogic acid with anamine, such as morpholine, with or without the presence of a base, suchas Et₃N, produced the morpholine addition product of gambogic acid(Scheme 7). Reaction of the piperidine amide of gambogic acid withN-methylpiperazine produced the N-methylpiperazine addition product ofthe amide (Scheme 8). Reduction of gambogic acid by NaBH₄ gave9,10-dihydro-12-hydroxygambogic acid, which may be oxidized byDess-Martin reagent to gave 9,10-dihydro-gambogic acid (Scheme 9).Alternatively, selective reduction of gambogic acid by L-selectride alsoproduced 9,10-dihydro-gambogic acid (Scheme 9). Reaction of gambogicacid with an alkylcuprate, such as cyclohexylcuprate, resulted in theaddition of the alkyl group to the 10-position, thereby producing10-cyclohexyl-gambogic acid (Scheme 10).

[0277] An important aspect of the present invention is the discoverythat compounds having Formula I-III are activators of caspases andinducers of apoptosis. Therefore, these compounds are expected to beuseful in a variety of clinical conditions in which there isuncontrolled cell growth and spread of abnormal cells, such as in thecase of cancer.

[0278] Another important aspect of the present invention is thediscovery that compounds having Formula I-III are potent and highlyefficacious activators of caspases and inducers of apoptosis in drugresistant cancer cells, such as breast and prostate cancer cells, whichenables these compounds to kill these drug resistant cancer cells. Incomparison, most standard anticancer drugs are not effective in killingdrug resistant cancer cells under the same conditions. Therefore,gambogic acid, its derivatives and analogs are expected to be useful forthe treatment of drug resistant cancer in animals.

[0279] The present invention includes a therapeutic method useful tomodulate in vivo apoptosis or in vivo neoplastic disease, comprisingadministering to a subject in need of such treatment an effective amountof a compound, or a pharmaceutically acceptable salt or prodrug of acompound of Formulae I-III, which functions as a caspase cascadeactivator and inducer of apoptosis.

[0280] The present invention also includes a therapeutic methodcomprising administering to an animal an effective amount of a compound,or a pharmaceutically acceptable salt or prodrug of said compound ofFormulae I-III, wherein said therapeutic method is useful to treatcancer, which is a group of diseases characterized by the uncontrolledgrowth and spread of abnormal cells. Such diseases include, but are notlimited to, Hodgkin's disease, non-Hodgkin's lymphomas, acute andchronic lymphocytic leukemias, multiple myeloma, neuroblastoma, breastcarcinomas, ovarian carcinomas, lung carcinomas, Wilms' tumor, cervicalcarcinomas, testicular carcinomas, soft-tissue sarcomas, chroniclymphocytic leukemia, primary macroglobulinemia, bladder carcinomas,chronic granulocytic leukemia, primary brain carcinomas, malignantmelanoma, small-cell lung carcinomas, stomach carcinomas, coloncarcinomas, malignant pancreatic insulinoma, malignant carcinoidcarcinomas, malignant melanomas, choriocarcinomas, mycosis fungoides,head and neck carcinomas, osteogenic sarcoma, pancreatic carcinomas,acute granulocytic leukemia, hairy cell leukemia, neuroblastoma,rhabdomyosarcoma, Kaposi's sarcoma, genitourinary carcinomas, thyroidcarcinomas, esophageal carcinomas, malignant hypercalcemia, cervicalhyperplasia, renal cell carcinomas, endometrial carcinomas, polycythemiavera, essential thrombocytosis, adrenal cortex carcinomas, skin cancer,and prostatic carcinomas.

[0281] In practicing the therapeutic methods, effective amounts ofcompositions containing therapeutically effective concentrations of thecompounds formulated for oral, intravenous, local and topicalapplication, for the treatment of neoplastic diseases and other diseasesin which caspase cascade mediated physiological responses areimplicated, are administered to an individual exhibiting the symptoms ofone or more of these disorders. The amounts are effective to ameliorateor eliminate one or more symptoms of the disorders. An effective amountof a compound for treating a particular disease is an amount that issufficient to ameliorate, or in some manner reduce, the symptomsassociated with the disease. Such amount may be administered as a singledosage or may be administered according to a regimen, whereby it iseffective. The amount may cure the disease but, typically, isadministered in order to ameliorate the disease. Typically, repeatedadministration is required to achieve the desired amelioration ofsymptoms.

[0282] In another embodiment, a pharmaceutical composition comprising acompound, or a pharmaceutically acceptable salt of said compound ofFormulae I-III, which functions as a caspase cascade activator andinducer of apoptosis in combination with a pharmaceutically acceptablevehicle is provided.

[0283] Another embodiment of the present invention is directed to acomposition effective to inhibit neoplasia comprising a compound, or apharmaceutically acceptable salt or prodrug of said compound of FormulaeI-III, which functions as a caspase cascade activator and inducer ofapoptosis, in combination with at least one known cancerchemotherapeutic agent, or a pharmaceutically acceptable salt of saidagent. Examples of known anti-cancer agents which can be used forcombination therapy include, but are not limited to, alkylating agentssuch as busulfan, cis-platin, mitomycin C, and carboplatin; antimitoticagents such as colchicine, vinblastine, paclitaxel, and docetaxel; topoI inhibitors such as camptothecin and topotecan; topo II inhibitors suchas doxorubicin and etoposide; RNA/DNA antimetabolites such as5-azacytidine, 5-fluorouracil and methotrexate; DNA antimetabolites suchas 5-fluoro-2′-deoxy-uridine, ara-C, hydroxyurea and thioguanine;antibodies such as Herceptin and Rituxan. Other known anti-cancer agentswhich can be used for combination therapy include melphalan,chlorambucil, cyclophosamide, ifosfamide, vincristine, mitoguazone,epirubicin, aclarubicin, bleomycin, mitoxantrone, elliptinium,fludarabine, octreotide, retinoic acid, tamoxifen and alanosine.

[0284] In practicing the methods of the present invention, the compoundof the invention may be administered together with at least one knownchemotherapeutic agent as part of a unitary pharmaceutical composition.Alternatively, the compound of the invention may be administered apartfrom the at least one known cancer chemotherapeutic agent. In thisembodiment, the compound of the invention and the at least one knowncancer chemotherapeutic agent are administered substantiallysimultaneously, i.e. the compounds are administered at the same time orone after the other, so long as the compounds reach therapeutic levelsin the blood.

[0285] Another embodiment of the present invention is directed to acomposition effective to inhibit neoplasia comprising a bioconjugate ofsaid compound of Formulae I-III, which functions as a caspase cascadeactivator and inducer of apoptosis, in bioconjugation with at least oneknown therapeutically useful antibody, such as Herceptin or Rituxan,growth factors such as DGF, NGF, cytokines such as IL-2, IL-4, or anymolecule that binds to the cell surface. The antibodies and othermolecules will deliver the compound of Formulae I-III to its target andmake it an effective anticancer agent. The bioconjugate could alsoenhance the anticancer effect of therapeutically useful antibodies, suchas Herceptin or Rituxan.

[0286] Similarly, another embodiment of the present invention isdirected to a composition effective to inhibit neoplasia comprising acompound, or a pharmaceutically acceptable salt or prodrug of saidcompound of Formulae I-III, which functions as a caspase cascadeactivator and inducer of apoptosis, in combination with radiationtherapy. In this embodiment, the compound of the invention may beadministered at the same time as the radiation therapy is administeredor at a different time.

[0287] Yet another embodiment of the present invention is directed to acomposition effective for post-surgical treatment of cancer, comprisinga compound, or a pharmaceutically acceptable salt or prodrug of saidcompound of Formulae I-III, which functions as a caspase cascadeactivator and inducer of apoptosis. The invention also relates to amethod of treating cancer by surgically removing the cancer and thentreating the animal with one of the pharmaceutical compositionsdescribed herein.

[0288] A wide range of immune mechanisms operate rapidly followingexposure to an infectious agent. Depending on the type of infection,rapid clonal expansion of the T and B lymphocytes occurs to combat theinfection. The elimination of the effector cells following an infectionis one of the major mechanisms maintaining immune homeostasis. Thisdeletion of reactive cells has been shown to be regulated by aphenomenon known as apoptosis. Autoimmune diseases have been recentlyidentified to occur as a consequence of deregulated cell death. Incertain autoimmune diseases, the immune system directs its powerfulcytotoxic effector mechanisms against specialized cells such asoligodendrocytes in multiple sclerosis, the beta cells of the pancreasin diabetes mellitus, and thyrocytes in Hashimoto's thyroiditis (Ohsako,S. & Elkon, K. B., Cell Death Differ 6(1):13-21 (1999)). Mutations ofthe gene encoding the lymphocyte apoptosis receptor Fas/APO-1/CD95 arereportedly associated with defective lymphocyte apoptosis and autoimmunelymphoproliferative syndrome (ALPS), which is characterized by chronic,histologically benign splenomegaly and generalized lymphadenopathy,hypergammaglobulinemia, and autoantibody formation. (Infante, A. J., etal., J. Pediatr. 133(5):629-633 (1998) and Vaishnaw, A. K., et al., J.Clin. Invest. 103(3):355-363 (1999)). It was reported thatoverexpression of Bcl-2, which is a member of the Bcl-2 gene family ofprogrammed cell death regulators with anti-apoptotic activity indeveloping B cells of transgenic mice, in the presence of T celldependent costimulatory signals, results in the generation of a modifiedB cell repertoire and in the production of pathogenic autoantibodies(Lopez-Hoyos, M., et al., Int. J. Mol. Med. 1(2):475-483 (1998)). It istherefore evident that many types of autoimmune diseases are caused bydefects of the apoptotic process. One treatment strategy for autoimmunediseases is to turn on apoptosis in the lymphocytes that are causing theautoimmune disease (O'Reilly, L. A. & Strasser, A., Inflamm Res48(1):5-21 (1999)).

[0289] Fas-Fas ligand (FasL) interaction is known to be required for themaintenance of immune homeostasis. Experimental autoimmune thyroiditis(EAT), characterized by autoreactive T and B cell responses and a markedlymphocytic infiltration of the thyroid, is a good model for the studyof the therapeutic effects of FasL. Batteux, F., et al., J. Immunol.162(1):603-608 (1999)) reported that the direct injection of DNAexpression vectors encoding FasL into the inflammed thyroid inhibitedthe development of lymphocytic infiltration of the thyroid. In addition,the death of infiltrating T cells was observed. These results show thatFasL expression on thyrocytes may have a curative effect on ongoing EATby inducing death of pathogenic autoreactive infiltrating T lymphocytes.

[0290] Bisindolylmaleimide VIII is known to potentiate Fas-mediatedapoptosis in human astrocytoma 1321N1 cells and in Molt-4T cells, andboth of which were resistant to apoptosis induced by anti-Fas antibodyin the absence of bisindolylmaleimide VIII. Potentiation of Fas-mediatedapoptosis by bisindolylmaleimide VIII was reported to be selective foractivated, rather than non-activated, T cells, and was Fas-dependent.Zhou T. et al. (Nat Med 5(1):42-8 (1999)) reported that administrationof bisindolylmaleimide VIII to rats during autoantigen stimulationprevented the development of symptoms of T cell-mediated autoimmunediseases in two models: the Lewis rat model of experimental allergicencephalitis and the Lewis adjuvant arthritis model. Therefore, theapplication of a Fas-dependent apoptosis enhancer such asbisindolylmaleimide VIII may be therapeutically useful for the moreeffective elimination of detrimental cells and inhibition of Tcell-mediated autoimmune diseases. Therefore, an effective amount of acompound, or a pharmaceutically acceptable salt or prodrug of thecompound of Formulae I-III, which functions as a caspase cascadeactivator and inducer of apoptosis, should be an effective treatment forautoimmune disease.

[0291] Psoriasis is a chronic skin disease which is characterized byscaly red patches. Psoralen plus ultraviolet A (PUVA) is a widely usedand effective treatment for psoriasis vulgaris. Coven, et al.,Photodermatol Photoimmunol Photomed 15(1):22-7 (1999), reported thatlymphocytes treated with psoralen 8-MOP or TMP plus UVA displayed DNAdegradation patterns typical of apoptotic cell death. Ozawa, et al., J.Exp. Med 189(4):711-718 (1999) reported that induction of T cellapoptosis could be the main mechanism by which 312-nm UVB resolvespsoriasis skin lesions. Low doses of methotrexate may be used to treatpsoriasis to restore a clinically normal skin. Heenen, et al., Arch.Dermatol. Res. 290(5):240-245 (1998), reported that low doses ofmethotrexate may induce apoptosis and this mode of action could explainthe reduction in epidermal hyperplasia during treatment of psoriasiswith methotrexate. Therefore, an effective amount of a compound, or apharmaceutically acceptable salt or prodrug of the compound of FormulaeI-III, which functions as a caspase cascade activator and inducer ofapoptosis, should be an effective treatment for psoriasis.

[0292] Synovial cell hyperplasia is a characteristic of patients withrheumatoid arthritis (RA). Excessive proliferation of RA synovial cellsas well as defects in synovial cell death may be responsible forsynovial cell hyperplasia Wakisaka, et al., Clin. Exp. Immunol.114(l):119-28 (1998), found that although RA synovial cells could dievia apoptosis through Fas/FasL pathway, apoptosis of synovial cells wasinhibited by proinflammatory cytokines present within the synovium. Thissuggested that inhibition of apoptosis by the proinflammatory cytokinesmay contribute to the outgrowth of synovial cells, and lead to pannusformation and the destruction of joints in patients with RA. Therefore,an effective amount of a compound, or a pharmaceutically acceptable saltor prodrug of the compound of Formulae I-III, which functions as acaspase cascade activator and inducer of apoptosis, should be aneffective treatment for RA.

[0293] There has been an accumulation of convincing evidence thatapoptosis plays a major role in promoting resolution of the acuteinflammatory response. Neutrophils are constitutively programmed toundergo apoptosis, thus limiting their pro-inflammatory potential andleading to rapid, specific, and non-phlogistic recognition bymacrophages and semi-professional phagocytes (Savill, J., J. Leukoc.Biol. 61(4):375-80 (1997)). Boirivant, et al., Gastroenterology116(3):557-65 (1999), reported that lamina propria T cells isolated fromareas of inflammation in Crohn's disease, ulcerative colitis, and otherinflammatory states manifest decreased CD2 pathway-induced apoptosis.Moreover, studies of cells from inflamed Crohn's disease tissue indicatethat this defect is accompanied by elevated Bcl-2 levels. Therefore, aneffective amount of a compound, or a pharmaceutically acceptable salt orprodrug of the compound of Formulae I-III, which functions as a caspasecascade activator and inducer of apoptosis, should be an effectivetreatment for inflammation.

[0294] Compositions within the scope of this invention include allcompositions wherein the compounds of the present invention arecontained in an amount which is effective to achieve its intendedpurpose. While individual needs vary, determination of optimal ranges ofeffective amounts of each component is within the skill of the art.Typically, the compounds may be administered to mammals, e.g. humans,orally at a dose of 0.0025 to 50 mg/kg, or an equivalent amount of thepharmaceutically acceptable salt thereof, per day, per kg of body weightof the mammal being treated for apoptosis-mediated disorders.Preferably, about 0.01 to about 10 mg/kg is orally administered to treator prevent such disorders. For intramuscular injection, the dose isgenerally about one-half of the oral dose. For example, a suitableintramuscular dose would be about 0.0025 to about 25 mg/kg, and mostpreferably, from about 0.01 to about 5 mg/kg. If a known cancerchemotherapeutic agent is also administered, it is administered in anamount with is effective to achieve its intended purpose. The amounts ofsuch known cancer chemotherapeutic agents effective for cancer are wellknown to those of ordinary skill in the art.

[0295] The unit oral dose may comprise from about 0.01 to about 50 mg,preferably about 0.1 to about 10 mg of the compound of the invention.The unit dose may be administered one or more times daily as one or moretablets each containing from about 0.1 to about 10, conveniently about0.25 to 50 mg of the compound or its solvates.

[0296] In a topical formulation, the compound may be present at aconcentration of about 0.01 to 100 mg per gram of carrier.

[0297] In addition to administering the compound as a raw chemical, thecompounds of the invention may be administered as part of apharmaceutical preparation containing suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the compounds into preparations which can beused pharmaceutically. Preferably, the preparations, particularly thosepreparations which can be administered orally and which can be used forthe preferred type of administration, such as tablets, dragees, andcapsules, and also preparations which can be administered rectally, suchas suppositories, as well as suitable solutions for administration byinjection or orally, contain from about 0.01 to 99 percent, preferablyfrom about 0.25 to 75 percent of active compound(s), together with theexcipient.

[0298] Also included within the scope of the present invention are thenon-toxic pharmaceutically acceptable salts of the compounds of thepresent invention. Acid addition salts are formed by mixing a solutionof the particular apoptosis inducers of the present invention with asolution of a pharmaceutically acceptable non-toxic acid such ashydrochloric acid, fumaric acid, maleic acid, succinic acid, aceticacid, citric acid, tartaric acid, carbonic acid, phosphoric acid, oxalicacid, and the like. Basic salts are formed by mixing a solution of theparticular apoptosis inducers of the present invention with a solutionof a pharmaceutically acceptable non-toxic base such as sodiumhydroxide, potassium hydroxide, choline hydroxide, sodium carbonate,Tris, N-methyl-glucamine and the like.

[0299] The pharmaceutical compositions of the invention may beadministered to any animal which may experience the beneficial effectsof the compounds of the invention. Foremost among such animals aremammals, e.g., humans and veterinary animals, although the invention isnot intended to be so limited.

[0300] The pharmaceutical compositions of the present invention may beadministered by any means that achieve their intended purpose. Forexample, administration may be by parenteral, subcutaneous, intravenous,intramuscular, intraperitoneal, transdermal, buccal, intrathecal,intracranial, intranasal or topical routes. Alternatively, orconcurrently, administration may be by the oral route. The dosageadministered will be dependent upon the age, health, and weight of therecipient, kind of concurrent treatment, if any, frequency of treatment,and the nature of the effect desired.

[0301] The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself known, for example, by means ofconventional mixing, granulating, dragee-making, dissolving, orlyophilizing processes. Thus, pharmaceutical preparations for oral usemay be obtained by combining the active compounds with solid excipients,optionally grinding the resulting mixture and processing the mixture ofgranules, after adding suitable auxiliaries, if desired or necessary, toobtain tablets or dragee cores.

[0302] Suitable excipients are, in particular, fillers such assaccharides, for example lactose or sucrose, mannitol or sorbitol,cellulose preparations and/or calcium phosphates, for example tricalciumphosphate or calcium hydrogen phosphate, as well as binders such asstarch paste, using, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, tragacanth, methyl cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/orpolyvinyl pyrrolidone. If desired, disintegrating agents may be addedsuch as the above-mentioned starches and also carboxymethyl-starch,cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a saltthereof, such as sodium alginate. Auxiliaries are, above all,flow-regulating agents and lubricants, for example, silica, talc,stearic acid or salts thereof, such as magnesium stearate or calciumstearate, and/or polyethylene glycol. Dragee cores are provided withsuitable coatings which, if desired, are resistant to gastric juices.For this purpose, concentrated saccharide solutions may be used, whichmay optionally contain gum arabic, talc, polyvinyl pyrrolidone,polyethylene glycol and/or titanium dioxide, lacquer solutions andsuitable organic solvents or solvent mixtures. In order to producecoatings resistant to gastric juices, solutions of suitable cellulosepreparations such as acetylcellulose phthalate orhydroxypropymethyl-cellulose phthalate, are used. Dye stuffs or pigmentsmay be added to the tablets or dragee coatings, for example, foridentification or in order to characterize combinations of activecompound doses.

[0303] Other pharmaceutical preparations which may be used orallyinclude push-fit capsules made of gelatin, as well as soft, sealedcapsules made of gelatin and a plasticizer such as glycerol or sorbitol.The push-fit capsules may contain the active compounds in the form ofgranules which may be mixed with fillers such as lactose, binders suchas starches, and/or lubricants such as talc or magnesium stearate and,optionally, stabilizers. In soft capsules, the active compounds arepreferably dissolved or suspended in suitable liquids, such as fattyoils, or liquid paraffin. In addition, stabilizers may be added.

[0304] Possible pharmaceutical preparations which may be used rectallyinclude, for example, suppositories, which consist of a combination ofone or more of the active compounds with a suppository base. Suitablesuppository bases are, for example, natural or synthetic triglycerides,or paraffin hydrocarbons. In addition, it is also possible to usegelatin rectal capsules which consist of a combination of the activecompounds with a base. Possible base materials include, for example,liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.

[0305] Suitable formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form, forexample, water-soluble salts and alkaline solutions. In addition,suspensions of the active compounds as appropriate oily injectionsuspensions may be administered. Suitable lipophilic solvents orvehicles include fatty oils, for example, sesame oil, or synthetic fattyacid esters, for example, ethyl oleate or triglycerides or polyethyleneglycol-400 (the compounds are soluble in PEG-400). Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension include, for example, sodium carboxymethyl cellulose,sorbitol, and/or dextran. Optionally, the suspension may also containstabilizers.

[0306] In accordance with one aspect of the present invention, compoundsof the invention are employed in topical and parenteral formulations andare used for the treatment of skin cancer.

[0307] The topical compositions of this invention are formulatedpreferably as oils, creams, lotions, ointments and the like, by choiceof appropriate carriers. Suitable carriers include vegetable or mineraloils, white petrolatum (white soft paraffin), branched chain fats oroils, animal fats and high molecular weight alcohol (greater than C₁₂).The preferred carriers are those in which the active ingredient issoluble. Emulsifiers, stabilizers, humectants and antioxidants may alsobe included as well as agents imparting color or fragrance, if desired.Additionally, transdermal penetration enhancers may be employed in thesetopical formulations. Examples of such enhancers are found in U.S. Pat.Nos. 3,989,816 and 4,444,762.

[0308] Creams are preferably formulated from a mixture of mineral oil,self-emulsifying beeswax and water in which mixture the activeingredient, dissolved in a small amount of an oil, such as almond oil,is admixed. A typical example of such a cream is one which includesabout 40 parts water, about 20 parts beeswax, about 40 parts mineral oiland about 1 part almond oil.

[0309] Ointments may be formulated by mixing a solution of the activeingredient in a vegetable oil, such as almond oil, with warm softparaffin and allowing the mixture to cool. A typical example of such anointment is one which includes about 30% almond oil and about 70% whitesoft paraffin by weight.

[0310] The following examples are illustrative, but not limiting, of themethod and compositions of the present invention. Other suitablemodifications and adaptations of the variety of conditions andparameters normally encountered in clinical therapy and which areobvious to those skilled in the art are within the spirit and scope ofthe invention.

EXAMPLE 1 Gambogic Acid

[0311] Procedure 1:

[0312] Step A.

[0313] The dry gamboge powder (140 g) was extracted with MeOH (3×600 mL)at room temperature for 1 week, after filtration, the solvent wasremoved under reduced pressure, gave crude extract (122 g) as yellowpowder.

[0314] Step B. Gambogic Acid Pyridine Salt.

[0315] The above crude extract (120 g) was dissolved in pyridine (120mL), then warm water (30 mL) was added to the stirred solution. Aftercooling to r.t., some precipitate was observed. Hexane (120 mL) wasadded to the mixture and the mixture was filtered and the solid waswashed with hexane and dried. The salt was purified by repeatedrecrystallization from ethanol and gave gambogic acid pyridine salt (7.5g); HPLC: 99%.

[0316] Step C. Gambogic Acid.

[0317] The gambogic acid pyridine salt (0.4 g) was dissolved in ether(25 mL) and shaken with aqeuous HCl (1N, 25 mL) for 1 h. The ethersolution was then washed with water (2×10 mL), dried and evaporated togive the title compound (345 mg); HPLC: 99%. ¹H NMR (CDCl₃): 12.66 (s,1H), 7.43 (d, J=6.9 Hz, 1H), 6.48 (d, J=10.2 Hz, 1H), 5.97 (t, J=7.5 Hz,1H), 5.26 (d, J=9.9 Hz, 1H), 4.91 (m, 2H), 3.37 (m, 1H), 3.24-2.98 (m,2H), 2.81 (d, J=6.6 Hz, 1H), 2.41 (d, J=9 Hz, 1H), 2.20 (m, J₁=8.4 Hz,J₂=5.1HZ, 1H), 1.88 (m, 1H), 1.63 (s, 3H), 1.60 (s, 3H), 1.58 (s, 3H),1.53 (s, 3H), 1.51 (s, 3H), 1.43 (s, 3H), 1.26 (s, 3H), 1.18 (s, 3H).MS: 627 (M−H).

[0318] Procedure 2:

[0319] The crude extract of gamboge (300 mg) was purified by repeatedcolumn chromatography (SiO₂, hexane-EtOAc gradient) using a Combi FlashSG 100 separation system, gave 18 mg of gambogic acid; HPLC: 94%, MS.627 (M−H).

EXAMPLE 2 Gambogenic Acid

[0320]

[0321] The crude extract of gamboge (300 mg) was purified as describedin Example 1, procedure 2, to give 3 mg of gambogenic acid; HPLC: 84%,MS. 629 (M−H).

EXAMPLE 3 Gambogenin

[0322]

[0323] The crude extract of gamboge (300 mg) was purified as describedin Example 1, procedure 2, to give 2 mg of gambogenin, HPLC: 71%. MS.613 (M−H).

EXAMPLE 4 Methyl Gambogate

[0324]

[0325] A mixture of gambogic acid (200 mg, 0.32 mmol), DMAP (78 mg, 0.64mmol), EDC (123 mg, 0.64 mmol) and methanol (102 mg, 3.2 mmol) in THF (5mL) was stirred at room temperature for 3 h. The solution was pouredinto water (10 mL) and was extracted with ethyl acetate (3×10 mL). Thecombined organic layer was dried and concentrated to give the crudeproduct, which was purified by chromatography (SiO₂, EtOAc/Hexane 1:5)to give the title compound (196 mg, 95%). ¹H NMR (CDCl₃): 12.85 (s, 1H),7.54 (d, J=6.9 Hz, 1H), 6.67 (d, J=10.5 Hz, 1H), 5.94 (t, J=6 Hz, 1H),5.43 (d, J=10.2 Hz, 1H), 5.05 (m, 2H), 3.49 (m, 1H), 3.43 (s, 3H),3.35-3.10 (s, 2H), 3.00 (t, J=7.2 Hz, 1H), 2.52 (d, J=10.2 Hz, 1H), 2.32(quar, J1=4.8 Hz, 1H), 2.02 (m, 1H), 1.74 (s, 3H), 1.69 (s, 3H),1.67-1.64 (m, 9H), 1.55 (s, 3H), 1.44 (s, 3H), 1.29 (s, 3H).

EXAMPLE 5 Gambogyl Piperidine

[0326]

[0327] A mixture of gambogic acid (200 mg, 0.32 mmol), DMAP (39 mg, 0.32mmol), EDC (123 mg, 0.64 mmol) and piperidine (54.2 mg, 0.64 mmol) inTHF (3 mL) was stirred at room temperature for 6 h. The solution waspoured into water (10 mL) and was extracted with ethyl acetate (3×10mL). The combined organic layer was dried and concentrated to give thecrude product, which was purified by chromatography (SiO₂, EtOAc/CH₂Cl₂1:8) to give the title compound (187 mg, 84%). ¹H NMR (CDCl₃): 12.87 (s,1H), 7.53 (d, J=6.9 Hz, 1H), 6.68 (d, J=10.2 Hz, 1H), 5.43 (d, J=10.5Hz, 1H), 5.40 (t, J=6 Hz, 1H), 5.05 (m, 2H), 3.54-3.33 (m, 2H), 3.28 (d,J=6.9 Hz, 1H), 3.11 (t, J=8.4 Hz, 1H), 2.50(d, J=9.6 Hz, 1H), 2.46-2.17(m, 3H), 2.00 (m, 1H), 1.75-1.72 (m, 5H), 1.68 (s, 2H), 1.65 (bs, 6H),1.58 (s, 3H), 1.56 (s, 3H), 1.43 (s, 3H), 1.25 (s, 3H).

EXAMPLE 6 Methyl-6-methoxy-gambogate

[0328]

[0329] A mixture of methyl gambogate (70 mg, 0.11 mmol), anhydrous K₂CO₃(0.5 g), methyl iodide (1 mL) in acetone (5 mL) was stirred at roomtemperature for 70 h. After evaporation to near dryness, water (30 mL)was added into the mixture and it was extracted with ethyl acetate (3×10mL). The combined organic layer was dried and concentrated to give thecrude product, which was purified by chromatography (SiO₂, EtOAc/Hexane1:4) to give the title compound (69 mg, 96%). MS. 657(M+H), 679 (M+Na⁻).¹H NMR (CDCl₃): 7.41 (d, J=6.9 Hz, 1H), 6.64 (d, J=9.9 Hz, 1H), 5.93 (m,J1=6.9 Hz, J2=0.9 Hz, 1H), 5.52 (d, J=10.2 Hz, 1H), 5.05 (m, 2H), 3.79(s, 3H), 3.40 (s, 3H), 3.45-3.18 (m, 3H), 2.95 (d, J=9.6 Hz, 1H), 2.26(m, 1H), 2.02 (m, 1H), 1.73 (s, 3H), 1.66 (d, 3H), 1.63 (bs, 6H), 1.52(s, 3H), 1.42 (s, 3H), 1.27(s, 3H).

EXAMPLE 7 6-Methoxy-gambogyl Piperidine

[0330]

[0331] The title compound was prepared by a procedure similar to that ofExample 6 from gambogyl piperidine and methyl iodide. MS: 710 (M+H), 732(M+Na⁺). ¹H NMR (CDCl₃): 7.40 (d, J=6.9 Hz, 1H), 6.64 (d, J=10.2 Hz,1H), 5.53 (d, J=10.2 Hz, 1H), 5.34 (m, 1H), 5.09 (t, 1H), 5.04 (t, 1H),3.80 (s, 3H), 3.52(m, 3H), 3.38-3.31 (m, 3H), 3.11 (t, 2H), 2.50-1.98(m, 5H), 1.73 (s, 3H), 1.70 (d, 3H), 1.65 (s, 6H), 1.63 (bs, 6H), 1.53(s, 3H), 1.42 (s, 3H), 1.22 (s, 3H).

EXAMPLE 8 10-Morpholinyl-gambogyl Morpholine

[0332]

[0333] A mixture of gambogic acid (100 mg, 0.16 mmol), DMAP (20 mg, 0.16mmol), EDC (67.4 mg, 0.35 mmol) and morpholine (30.6 mg, 0.35 mmol) inTHF (3 mL) was stirred at room temperature for 6 h. The solution waspoured into water (10 mL) and was extracted with ethyl acetate (3×10mL). The combined organic layer was dried and concentrated to give thecrude product, which was purified by chromatography (SiO₂, EtOAc/CH₂Cl₂1:1) to give the title compound (87 mg, 69%). MS: 785 (M+H), 807 (M+H⁻).¹H NMR (CDCl₃): 11.94 (s, 1H), 6.63 (d, J=7.5 Hz, 1H), 5.96 (m, 1H),5.43 (d, J=10.2 Hz, 1H), 5.08 (m, 2H), 3.80 (m, 1H), 3.70-3.12 (m, 12H),2.80-2.36 (m, 7H), 2.05 (m, 1H), 1.95 (m, 1H), 1.87 (s, 3H), 1.73 (bs,3H), 1.64 (bs, 6H), 1.55 (s, 3H), 1.45 (m, 1H), 1.32 (s, 3H), 1.28 (s,3H), 1.24 (3H), 1.07 (s, 3H).

EXAMPLE 9 Gambogyl (b 4-Methylpiperazine)

[0334]

[0335] A mixture of gambogic acid (93 mg, 0.15 mmol), DMAP (22 mg, 0.18mmol), EDC (34 mg, 0.18 mmol) and N-methyl piperazine (15 mg, 0.15 mmol)in THF (5 mL) was stirred at room temperature for 5 h. The solution waspoured into water (50 mL) and was extracted with ethyl acetate (3×10mL). The combined organic layer was dried and concentrated to give crudeproduct, which was purified by chromatography (SiO₂, EtOAc/MeOH 12:1) togive the title compound (35 mg, 33%). MS: 709 (M−H), 711 (M+H), 733(M+Na⁺). ¹H NMR (CDCl₃): 12.85 (s, 1H), 7.52 (d, J=6.6 Hz, 1H), 6.66 (d,J=9.9 Hz, 1H), 5.42 (t, J=10.5 Hz, 1H), 5.05 (m, 2H), 3.62 (m, 1H), 3.40(m, 2H), 3.28-3.17 (m, 4H), 2.50-1.98 (m, 7H), 2.23 (s, 3H), 1.72 (bs,6H), 1.63 (bs, 6H), 1.53 (bs, 6H), 1.41 (s, 3H), 1.23 (s, 3H).

EXAMPLE 10 10-Morpholinyl-gambogyl Piperidine

[0336]

[0337] A solution of gambogyl piperidine (50 mg, 0.071 mmol) andmorpholine (0.3 mL) in THF (3 mL) was stirred for 48 h. It wasevaporated and the crude product was purified through chromatography toyield the title compound (48 mg, 86%). ¹H NMR (CDCl₃) 11.94 (s, 1H),6.66 (d, J=9.9 Hz, 1H), 5.92 (t, 1H), 5.44 (d, J=10.2 Hz, 1H), 5.06 (m,1H), 3.72-3.12 (m, 12H), 2.80 (m, 3H), 2.60-2.40 (m, 4H), 2.06 (m, 2H),1.88 (s, 3H), 1.75 (s, 3H), 1.66 (s, 3H), 1.65 (s, 3H), 1.57 (s, 3H),1.55 (m, 2H), 1.34 (s, 3H), 1.32 (s, 3H), 1.22 (s, 2H), 1.11 (s, 3H).

EXAMPLE 11 10-(4-Methylpiperazinyl)-gambogyl Piperidine

[0338]

[0339] The title compound was prepared from gambogyl piperidine andN-methylpiperazine by a procedure similar to that of Example 10. MS. 797(M+H), 819 (M+Na), 835 (M+K), 795 (M−H). ¹H NMR (CDCl₃) 12.01 (s, H),6.66 (d, J=9.9 Hz, 1H), 5.94 (t, 1H), 5.44 (d, J=10.2 Hz, 1H), 5.12-5.10(m, 2H), 3.80 (d, 1H), 3.52 (d, 1H), 3.38-3.12 (m, 5H), 2.78-2.26 (m,6H), 2.24 (s, 3H), 2.12-2.04 (m, 2H),), 1.89 (s, 3H), 1.75 (s, 3H), 1.66(s, 6H), 1.57 (s, 3H), 1.55 (m, 2H), 1.34 (s, 3H), 1.32 (s, 3H), 1.11(s, 3H).

EXAMPLE 12 N-(2-Gambogylamidoethyl)biotinamide

[0340]

[0341] The title compound was prepared by a procedure similar to that ofExample 9 from gambogic acid and N-(2-aminoethyl)biotinamide. MS: 919(M+Na), 897 (M+H), 895 (M−H). ¹H NMR (CDCl₃): 12.9, 12.78 (1H),7.60-7.57 (m, 1H), 6.90 (m, 1H), 6.78 (m, 1H), 6.70-6.62 (m, 1H),5.48(d, J=9.9 Hz, 1H), 5.42 (m, 1H), 5.30 (m, 1H), 5.08 (m, 2H), 4.66(s,1H), 4.49 (m, 1H), 4.33 (m,1H), 3.58-3.40 (m, 2H), 3.38-3.10 (m, 5H),3.16-2.88 (m, 1H), 2.80-2.52 (m, 2H), 2,40-1.92 (m, 6H), 1.78 (bs, 3H),1.74 (bs, 2H), 1.73 (bs, 3H), 1.69 (bs, 3H), 1.65 (bs, 6H), 1.55 (bs,3H), 1.50-1.20 (m, 13H), 1.2-0.88 (m, 4H).

EXAMPLE 13 10-(4-Methylpiperazinyl)gambogic Acid

[0342]

[0343] A solution of gambogic acid (35 mg, 0.056 mmol) andN-methylpiperazine (0.5 mL) in THF (4 mL) was stirred for 24 h, thenanother portion of N-methylpiperazine (0.5 mL) was added and stirred for48 h. The solution was diluted with EtOAc (30 mL) and washed withaqueous NH₄Cl (3×30 mL). After concentration, the mixture was dissolvedin ethyl ether (15 mL) and washed with 0.1 N HCl. After concentration,the residue was washed with hexane four times to gave the title compound(9 mg, 20%). ¹H NMR (CDCl₃) 11.8 (s, 1H), 6.64 (d, J=9.9 Hz, 1H),6.57(t, 1H), 5.45 (d, J=10.5 Hz, 1H), 5.09 (t, 1H), 3.51 (bs, 1H),3.30-2.70 (m, 11H), 2.81 (s, 3H), 2.51 (d, J=8.4 Hz, 2H), 2.12-2.02 (m,2H), 1.96 (s, 3H), 1.73 (s, 3H), 1.66 (s, 3H), 1.63 (s, 3H), 1.56 (s,3H), 1.35 (s, 6H), 1.26 (m, 2H), 1.11 (s, 3H), 0.88 (m, 2H).

EXAMPLE 14 10-Piperidyl-gambogyl Piperidine

[0344]

[0345] A mixture of gambogic acid (460 mg, 0.73 mmol), EDCI (166 mg,0.87 mmol), DMAP (47 mg, 0.38 mmol) and piperidine (75 μL, 0.76 mmol) inTHF (5 mL) was stirred at room temperature for 40 h. It was diluted with1:1 hexane/EtOAc (80 mL), washed with water and brine, dried over Na₂SO₄and concentrated in vacuo. The residue was purified by chromatography(3:2 hexane/EtOAc) to yield the title compound as a pale yellow solid(40 mg, 0.051 mmol, 7%) and gambogyl 1′-piperidine (220 mg, 0.32 mmol).¹H NMR (CDCl₃): 12.05 (s, 1H), 6.66 (d, J=10.0 Hz, 1H), 5.95 (t, J=6.3Hz, 1H), 5.44 (d, J=10.0 Hz, 1H), 5.12-5.03 (m, 2H), 3.75-3.10 (m, 9H),2.76-2.66 (m, 3H), 2.49 (d, J=8.4, 2H), 2.34(m, 2H), 2.08 (m, 3H), 1.89(s, 3H), 1.75 (s, 3H), 1.66 (d, 3H), 1.61-1.23 (m, 15H), 1.33 (s, 3H),1.31 (s, 3H), 1.27(s, 3H), 1.26 (s, 3H).

EXAMPLE 15 10-Piperidinyl-gambogic Acid

[0346]

[0347] Gambogic acid (10 mg, 0.016 mmol) in piperidine (0.5 mL) wasstirred at room temperature for 40 h. The solvent was removed in vacuo.The residue was diluted with 1:2 hexane/EtOAc (50 mL), washed withsaturated ammonium chloride aqueous solution followed by brine, driedover Na₂SO₄ and concentrated in vacuo. The residue was purified bychromatography (3:2 hexane/EtOAc) to yield one of the diasteromers ofthe title compound (A, 3 mg, 0.004 mmol, 26%) and the other diastereomer(B, 1 mg, 0.001 mmol, 6%). ¹H NMR (CDCl₃): diastereomer A: 12.00 (s,1H), 6.66 (d, J=9.9 Hz, 1H), 6.52 (t, J=6.9 Hz, 1H), 5.46 (d, J=9.9 Hz,1H), 5.12-5.05 (m, 2H), 3.32-3.04 (m, 6H), 2.81 (t, J=4.5, 1H),2.55-2.43 (m, 3H), 2.33(m, 2H), 2.12-1.91 (m, 3H), 1.98 (s, 3H), 1.74(s, 3H), 1.66 (s, 3H), 1.63 (s, 3H), 1.35 (s, 6H), 1.50-1.28 (m, 6H),1.14 (s, 3H); diastereomer B: 12.00 (s, 1H), 7.37 (t, J=6.3 Hz, 1H),6.66 (d, J=10.0 Hz, 1H), 5.46 (d, J=10.0 Hz, 1H), 5.12-5.02 (m, 2H),3.35-3.18 (m, 3H), 3.11 (s, 1H), 2.91-2.79 (m, 3H), 2.56-2.48 (m, 3H),2.33(m, 2H), 2.12-1.94 (m, 5H), 1.87 (s, 3H), 1.74 (s, 3H), 1.66 (s,3H), 1.63 (s, 3H), 1.40 (s, 3H), 1.34 (s, 3H), 1.50-1.28 (m, 6H), 1.13(s, 3H).

EXAMPLE 16 9,10-Dihydro-12-hydroxygambogic Acid

[0348]

[0349] To a solution of gambogic acid (14 mg, 0.022 mmol) in methanol (2mL) was added NaBH₄ (22 mg, 0.58 mmol) at 0° C. The mixture was stirredfor 3 h and the cooling bath was allowed to slowly warm to roomtemperature. Acetone (0.5 mL) was added to the mixture and it wasstirred for 30 min., acidified with 2 N HCl to pH 6, diluted with EtOAc(40 mL), washed with water (3 times) and brine, dried over Na₂SO₄ andconcentrated in vacuo. The residue was purified by chromatography (9:1EtOAc/MeOH) to give the title compound as an oil (9 mg, 0.014 mmol,66%). ¹H NMR (CDCl₃): 12.02 (s, 1H), 6.66 (d, J=10.2 Hz, 1H), 6.33 (t,J=7.2, 1H), 5.45 (d, J=10.2 Hz, 1H), 5.12-5.05 (m, 2H), 3.71 (s, 1H),3.26-3.11 (m, 3H), 3.02-2.94 (m, 2H), 2.56-2.49 (m, 1H), 2.36 (d, J=9.6,1H), 2.12-2.04 (m, 3H), 1.99 (s, 3H), 1.76 (m, 1H), 1.73 (s, 3H), 1.66(s, 3H), 1.63 (s, 3H), 1.60-1.30 (m, 5H), 1.42 (s, 3H), 1.39 (s, 3H),1.35 (s, 3H).

EXAMPLE 17 Methyl-10-Morpholinyl-gambogate

[0350]

[0351] To a solution of methyl gambogate (50 mg, 0.078 mmol) in THF (2mL) was added morpholine (70 L, 0.80 mmol). The mixture was stirred atroom temperature for 17 h, diluted with 1:1 hexane/EtOAc (100 mL),washed with water (3 times) and brine, dried over Na₂SO₄ andconcentrated in vacuo to yield the title compound as light yellow solid(52 mg, 0.071 mmol, 91%). ¹H NMR (CDCl₃): 11.98 (s, 1H), 6.66 (d, J=10.2Hz, 1H), 6.62 (t, J=6.6 Hz, 1H), 5.46 (d, J=10.2 Hz, 1H), 5.12-5.00 (m,2H), 3.68 (s, 3H), 3.74-3.55 (m, 4H), 3.43-3.14 (m, 6H), 2.77 (m, 1H),2.59-2.40 (m, 5H), 2.07(m, 1H), 1.95 (s, 3H), 1.74 (s, 3H), 1.66 (s,3H), 1.63 (s, 3H), 1.36 (s, 3H), 1.35 (s, 3H), 1.14 (s, 3H).

EXAMPLE 18 Isogambogic Acid

[0352]

[0353] The crude extract of gamboge (300 mg) was purified as describedin Example 1, procedure 2, to give 2 mg of isogambogic acid; MS. 627(M−H).

EXAMPLE 19 Morellic Acid

[0354]

[0355] The crude extract of gamboge (300 mg) was purified as describedin Example 1, procedure 2, to give 2 mg of morellic acid; MS. 559 (M−H).

EXAMPLE 20 10-Cyclohexylgambogic Acid

[0356]

[0357] To a solution of gambogic acid (80 mg, 0.13 mmol) in THF (5 mL)was added a solution of cyclohexylcuprate (1.2 mmol) in THF preparedfrom cyclohexylmagnesium chloride and CuI at 0° C. The mixture wasstirred for 2 h and the cooling bath was allowed to slowly warm to roomtemperature. The reaction was quenched with 2 N HCl and diluted with 1:1hexane/EtOAc (80 mL). The resulting mixture was washed with water andbrine, dried over Na₂SO₄ and concentrated in vacuo. The residue waspurified by chromatography (3:1 hexane/EtOAc) to give the title compoundas an oily solid (9 mg, 0.013 mmol, 10%). ¹H NMR (CDCl₃): 6.61 (d,J=10.2, 1H), 6.14 (t, J=6.0, 1H), 5.39 (d, J=10.2, 1H), 5.20 (t,J=6.6,1H), 5.06 (t, J=7.2, 1H), 3.64 (m, 1H), 3.35-3.10 (m, 3H), 2.82(br s, 2H), 2.67-2.61 (m, 2H), 1.76(s, 3H), 1.72 (s, 3H), 1.68 (s, 3H),1.66 (s, 3H), 1.56 (s, 3H), 1.44 (s, 3H), 1.94-1.25 (m, 15H).

EXAMPLE 21 10-Methylgambogic Acid

[0358]

[0359] The title compound was prepared by a procedure similar to that ofExample 20 from gambogic acid and methylcuprate and was isolated as anoil. ¹H NMR (CDCl₃): 6.58 (d, J=10.2, 1H), 6.12 (t, J=6.6, 1H), 5.36 (d,J=9.9, 1H), 5.15 (t, J=6.6, 1H), 5.04 (t, J=7.2, 1H), 3.32-3.10 (m, 2H),2.93 (d, J=8.4, 1H), 2.84 (d, J=6.3, 2H), 2.62 (d, J=7.6, 1H), 2.40 (t,J=7.2, 1H), 2.25 (d, J=4.8, 1H), 2.08-1.92 (m, 4H), 1.73(s, 3H), 1.68(s, 3H), 1.66 (s, 6H), 1.64 (s, 3H), 1.54 (s, 3H), 1.41 (s, 3H), 1.35(d, J=6.9, 3H), 1.23 (s, 1H).

EXAMPLE 22 10-Dihydrogambogic Acid

[0360]

[0361] To a solution of gambogic acid (17 mg, 0.027 mmol) inmethylenechloride (2 mL) was added L-selectride solution in THF (1.0 mL,0.5 mmol) dropwise at −78° C. After 30 min of stirring, the reaction wasquenched with 1 mL of 2 N HCl. The mixture was then allowed to warm toroom temperature and was diluted with 1:1 hexane/EtOAc (50 mL). Theresulting mixture was washed with water and brine, dried over Na₂SO₄ andconcentrated in vacuo. The residue was purified by chromatography (2:3hexane/EtOAc/) to give the title compound as an oil (0.6 mg, 0.001 mmol,4%). ¹H NMR (CDCl₃): 11.96 (s, 1H), 6.67 (d, J=10.2, 1H), 6.54 (t,J=6.6, 1H), 5.46 (d, J=10.2, 1H), 5.13-5.05 (m, 2H), 3.33-3.16 (m, 3H),2.85 (d, J=13.8, 1H), 2.60 (d, J=8.7, 1H), 2.43 (s, 1H), 2.08 (m, 1H),1.97 (s, 3H), 1.74 (s, 3H), 1.67 (s, 6H), 1.64 (s, 3H), 1.57 (s, 3H),1.37 (s, 3H), 1.36 (d, J=6.9, 3H), 1.31-1.22 (m, 5H), 1.14 (s, 3H).

EXAMPLE 23 Gambogyl (2-(4-Morpholinyl)ethylamine)

[0362]

[0363] The title compound was prepared as described in Example 5 fromgambogic acid and 2-(4-morpholinyl)ethylamine and isolated as a yellowsolid (75 mg, 0.10 mmol, 56%). ¹H NMR (CDCl₃): 12.86 (s, 1H), 7.54 (d,J=6.6, 1H), 6.68 (d, J=10.2, 1H), 6.56 (t, J=5.1, 1H), 5.46 (d, J=10.2,1H), 5.28 (d, J=7.5, 1H), 5.05 (br s, 1H), 3.68 (t, J=4.2, 4H), 3.47 (m,1H), 3.71-3.17 (m, 4H), 2.68 (t, J=6.6, 2H), 2.54 (d, J=9.6, 1H),2.48-2.44 (m, 6H), 2.36-2.30 (m, 1H), 2.01-2.00 (m, 3H), 1.74 (s, 6H),1.67 (s, 3H), 1.65 (s, 6H), 1.61 (s, 3H), 1.44 (s, 3H), 1.28 (s, 3H).

EXAMPLE 24 9,10-Epoxygambogic Acid

[0364]

[0365] To a solution of gambogic acid (52 mg, 0.08 mmol) in methanol (2mL) was added 2 N NaOH (0.5 mL, 1.0 mmol), followed by 35% H₂O₂ (0.2 mL,2.1 mmol) at room temperature. The mixture was stirred at roomtemperature for 10 min, diluted with 1:1 hexane/EtOAc (50 mL), washedwith water, 2 N HCl and brine, dried over Na₂SO₄ and concentrated invacuo. The residue was purified by chromatography (1:2 hexane/EtOAc) toyield the title compound as an oil (2.2 mg, 0.003 mmol, 4%). ¹H NMR(CDCl₃): 11.92 (s, 1H), 6.66 (d, J=10.2, 1H), 6.51 (t, J=6.9, 1H), 5.46(d, J=9.9, 1H), 5.09-5.04 (m, 2H), 4.35 (d, J=3.9, 1H), 3.32 (s, 2H),3.27-2.99 (m, 4H), 2.85 (t, J=4.8, 1H), 2.51 (d, J=8.7, 1H), 2.07 (m,1H), 1.97 (s, 3H), 1.74 (s, 3H), 1.66 (s, 3H), 1.63 (s, 3H), 1.56 (s,3H), 1.36 (s, 3H), 1.15 (s, 3H).

EXAMPLE 25 Gambogyl (4-(2-Pyridyl)piperazine) and10-[4-(2-Pyridyl)piperazinyl]gambogyl(4-(2-Pyridyl)piperazine)

[0366]

[0367] A mixture of gambogic acid (230 mg, 0.37 mmol),1-(2-pyridyl)piperazine (75 μL, 0.46 mmol), and EDC (77 mg, 0.40 mmol)in DMF (3 mL) was stirred at room temperature, overnight. The mixturewas diluted with 1:1 hexane/EtOAc (90 mL), washed with water and brine,dried over Na₂SO₄ and concentrated in vacuo. The residue was purified bychromatography (3:2 hexane/EtOAc) to yield 10 mg of gambogyl(4-(2-pyridyl)piperazine) as a yellow solid. ¹H NMR (CDCl₃): 12.87 (s,1H), 8.19 (m, 1H), 7.52 (d, J=6.9, 1H), 6.68-6.62 (m, 2H), 6.43 (d,J=9.9, 1H), 5.06 (br s, 2H), 3.76-3.27 (m, 11H), 2.52-2.00 (m, 6H), 1.76(s, 3H), 1.73 (s, 3H), 1.68 (s, 3H), 1.65 (s, 6H), 1.56 (s, 3H), 1.42(s, 3H), 1.26 (s, 3H); and 31 mg of10-[4-(2-pyridyl)piperazinyl]gambogyl(4-(2-pyridyl)piperazine) as ayellow solid. ¹H NMR (CDCl₃): 11.99 (s, 1H), 8.17 (d, J=4.8, 2H), 7.45(t, J=7.5, 2H), 6.68-6.52 (m, 5H), 6.00 (t, J=6.6, 1H), 5.44 (d, J=10.2,1H), 5.11-5.07 (m, 2H), 3.90-3.13 (m, 15H), 2.83-2.51 (m, 8H), 2.07 (m,2H), 1.90 (s, 3H), 1.73 (s, 3H), 1.65 (s, 6H), 1.57 (s, 3H), 1.35 (s,3H), 1.32 (s, 3H), 1.12 (s, 3H);

EXAMPLE 26 6-Acetyl-gambogic Acid

[0368]

[0369] A mixture of gambogic acid (154 mg, 0.24 mmol) and Ac₂O (0.3 mL,3.2 mmol) in pyridine (3 mL) was stirred at room temperature for fourdays. The mixture was diluted with 1:1 hexane/EtOAc (80 mL), washed withwater, 2 N HCl and brine, dried over Na₂SO₄ and concentrated in vacuo.The residue was purified by chromatography (1:2 hexane/EtOAc) to yieldthe title compound as a yellow solid (47 mg, 0.07 mmol, 29%). ¹H NMR(CDCl₃): 7.44 (d, J=6.9, 1H), 6.66 (t, J=6.6, 1H), 6.40 (d, J=10.2, 1H),5.60 (d, J=10.5, 1H), 5.13 (t, J=6.9, 1H), 5.04 (t, J=6.9, 1H), 3.46 (m,1H), 3.34 (d, J=6.9, 1H), 2.67-2.50 (m, 3H), 2.39 (s, 3H), 2.33-2.27 (m,1H), 2.08-1.98 (m, 2H), 1.73 (s, 3H), 1.71 (s, 3H), 1.65 (s, 6H), 1.54(s, 3H), 1.40 (s, 3H), 1.6 (s, 3H), 1.29 (s, 3H).

EXAMPLE 27 10-[4-(2-Pyridyl)piperazinyl]gambogic Acid

[0370]

[0371] A mixture of pyridinium gambogate (228 mg, 0.32 mmol) and1-(2-pyridyl)piperazine (289 mg, 1.8 mmol) in THF (3 mL) was stirred atroom temperature, overnight. The mixture was diluted with 1:1hexane/EtOAc (80 mL), washed with water, 2 N HCl and brine, dried overNa₂SO₄ and concentrated in vacuo to yield the title compound as a yellowsolid (143 mg, 0.16 mmol, 50%). ¹H NMR (CDCl₃): 11.97 (s, 1H), 8.15 (m,1H), 7.45 (m, 1H), 6.68-6.54 (m, 4H), 5.46 (d, J=9.9, 1H), 4.12-5.02 (m,2H), 3.40-3.08 (m, 10H), 2.83 (t, J=4.5, 1H), 2.71-2.67 (m, 2H),2.57-2.52 (m, 3H), 1.95 (s, 3H), 1.74 (s, 3H), 1.66(s, 3H), 1.63 (s,3H), 1.57 (s, 3H), 1.37 (s, 6H), 1.16 (s, 3H).

EXAMPLE 28 N-Hydroxysuccinimidyl Gambogate

[0372]

[0373] A mixture of gambogic acid (600 mg, 0.96 mmol),N-hydroxysuccinimide (221 mg, 1.92 mmol), DCC (296.6 mg, 1.44 mmol) indichloromethane (20 mL) was stirred for 2 h. It was evaporated todryness and the residue was dissolved in ethyl acetate (50 mL) andwashed with water (50 mL×3). The organic layer was dried andconcentrated to give crude product, which was purified by flash columnchromatography (SiO₂, EtOAc/hexane 1:3) to give the title compound (530mg, 76%). ¹H NMR (CDCl₃): 12.85 (s, 1H), 7.55 (d, J=6.9 Hz, 1H), 6.67(d, J=10.2 Hz, 1H), 6.62 (t, J=6.9 Hz, 1H), 5.44 (d, J=9.9 Hz, 1H), 5.06(m, 2H), 3.46 (m, 1H), 3.38-3.12 (m, 2H), 2.84-2.76 (m, 4H), 2.54 (d,1H), 2.30 (m, 1H), 2.04 (m, 1H), 1.94 (s, 3H), 1.74(s, 3H), 1.72 (s,3H), 1.66 (s, 3H), 1.63 (s, 3H), 1.56 (s, 3H), (bs, 6H), 1.43 (s, 3H),1.29 (s, 3H).

EXAMPLE 29 8-(Gambogylamido)octanoic Acid

[0374]

[0375] A solution of 8-aminooctanoic acid (3.07 mg, 0.019 mmol),N-hydroxysuccinimidyl gambogate (14 mg, 0.019 mmol), triethylamine (0.15mL) in anhydrous DMSO (3 mL) was stirred overnight. It was diluted withwater and extracted with ethyl acetate (3×10 mL). The combined organiclayer was dried and concentrated to give crude product, which waspurified by column chromatography (SiO₂, EtOAc/MeOH 10:1) to give thetitle compound (11 mg, 67%). ¹H NMR (CDCl₃): 12.80 (bs, 1H), 7.58 (bs,1H), 6.64 (d, J=9.3 Hz, 1H), 5.50-5.00 (m, 4H), 3.54 (bs, 1H), 3.32-3.00(m, 3H), 3.50-2.42 (m, 4H), 1.75 (s, 3H), 1.72 (s, 3H), 1.70 (s, 3H).MS. 792 (M+Na⁺), 768 (M−H).

EXAMPLE 30 6-(Gambogylamido)hexanoic Acid

[0376]

[0377] The title compound was prepared by a procedure similar to that ofExample 29. ¹H NMR (CDCl₃): 12.70 (bs, 1H), 7.58 (bs, 1H), 6.62 (bs,1H), 5.40 (bs, 1H), 5.20 (bs, 1H), 5.00 (bs, 2H), 3.60-3.00 (m, 4H),3.50-2.42 (m, 4H), 1.74 (s, 3H), 1.72 (s, 3H), 1.69 (s, 3H). MS. 764(M+Na⁺), 740 (M−H).

EXAMPLE 31 12-(Gambogylamido)dodecanoic Acid

[0378]

[0379] The title compound was prepared by a procedure similar to that ofExample 29. ¹H NMR (CDCl₃): 12.7 (bs, 1H), 7.48 (d, 1H), 6.64 (d, J=10.5Hz, 1H), 5.50-5.00 (m, 6H), 3.50 (bs, 1H), 3.40-3.00 (m, 3H), 2.80-1.92(m, 6H). 1.75 (s, 3H), 1.73 (s, 3H), 1.71 (s, 3H), 1.56 (s, 3H). MS. 849(M+Na⁺), 825 (M−1).

EXAMPLE 32 N-Hydroxysuccinimidyl-8-(Gambogylamido)octanoate

[0380]

[0381] The title compound was prepared by a procedure similar to that ofExample 28. ¹H NMR (CDCl₃): 12.70 (s, 1H), 7.55 and 7.51 (d, J=6.9 Hz,1H), 6.65 and 6.64 (d, J=10.2 Hz, 1H), 5.50-5.00 (m, 4H), 4.12 (d, 2H),3.49 (m, 2H), 3.30 (t, J=6.6 Hz, 1H), 3.19 (m, 3H), 2.85 (s, 4H),2.70-2.50 (m, 3H), 2.04 (m, 1H), 1.75 (bs, 3H), 1.74(s, 3H), 1.72 (s,3H), 1.70 (s, 3H), 1.56 (s, 3H), 1.42 (s, 3H), 1.33 (bs, 3H), 1.30 (bs,3H). MS. 889 (M+Na⁺), 865 (M−H).

EXAMPLE 33 N-Hydroxysuccinimidyl-6-(Gambogylamido)hexanoate

[0382]

[0383] The title compound was prepared by a procedure similar to that ofExample 28. ¹H NMR (CDCl₃): 12.70 (s, 1H), 7.56 and 7.52 (d, J=6.9 Hz,1H), 6.69 and 6.65(d, J=10.2 Hz, 1H), 6.59 (t, 1H), 5.60-5.00 (m, 4H),4.10 (m, 2H), 3.60-3.12 (m, 6H), 2.85 (s, 4H), 2.70-2.50 (m, 3H), 2.35(m, 1H), 2.04 (m, 1H), 1.90 (m, 4H), 1.73(s, 3H), 1.72 (s, 3H), 1.69 (s,3H), 1.56 (bs, 6H), 1.44(s, 3H), 1.33(s, 3H), 1.29(s, 3H). MS. 861(M+Na₊), 837 (M−H).

EXAMPLE 34 N-Hydroxysuccinimidyl-12-(Gambogylamido)dodecanoate

[0384]

[0385] The title compound was prepared by a procedure similar to that ofExample 28. ¹H NMR (CDCl₃): 12.70 (s, 1H), 7.55 and 7.51 (d, J=7.2 Hz,1H), 6.66 and 6.64(d, J=9.9 Hz, 1H), 5.47(d, J=10.5 Hz, 1H), 5.46-5.10(m, 3H), 4.08 (m, 4H), 3.56-3.40 (m, 4H), 3.18 (m, 2H), 2.60 (t, 1H),2.83 (s, 4H). MS. 946 (M+Na⁺), 922 (M−H).

EXAMPLE 35 10-Methoxy-gambogyl Piperidine

[0386]

[0387] To a solution of gambogyl piperidine (30 mg, 0.043 mmol) inmethanol (4 mL) was added sodium methoxide (4.6 mg, 0.086 mmol) and itwas stirred at room temperature for 3 h. The reaction was poured intoice water (20 mL), and extracted with ethyl acetate (3×10 mL). Theorganic extract was dried and concentrated to give crude product, whichwas purified by chromatography to give the title compound (18 mg, 58%).MS. 726 (M−H⁺), 750 (M+Na⁻). ¹H NMR(CDCl₃): 11.98 (s, 1H), 6.65 (d,J=10.2 Hz, 1H), 5.77 (t, J=6.6 Hz, 1H), 5.43 (d, J=10.2 Hz, 1H), 5.07(m, 2H), 4.33 (d, 1H), 3.60-3.15 (m, 3H), 3.31 (s, 3H), 2.80-2.40 (m,3H), 1.87 (s, 3H), 1.66 (s, 3H), 1.60 (s, 3H), 1.36 (s, 3H), 1.31 (s,3H), 1.28 (s, 3H), 1.23 (s, 3H), 1.11 (s, 3H).

EXAMPLE 36 Gambogyl (2-Dimethylaminoethylamine)

[0388]

[0389] The title compound was prepared by a procedure similar to that ofExample 29. MS. 697 (M−H⁻), 699 (M+H⁺). ¹H NMR (CDCl₃): 12.90 (bs, 1H),7.54 (d, J=6.9 Hz, 1H), 6.68 (d, J=9.6 Hz, 1H), 6.52 (t, 1H), 5.45 (d,J=10.2 Hz, 1H), 5.37 (dt, J₁=8.4 Hz, J₂=1.5 Hz, 1H), 5.05 (m, 2H),3.50-3.10 (m,3H), 2.21 (s, 6H), 1.76 (s, 3H), 1.75 (s, 3H), 1.69 (s,3H), 1.65 (s, 3H), 1.64 (s, 3H), 1.56 (s, 3H), 1.44 (s, 3H), 1.29 (s,3H).

[0390] The following compounds (Examples 37-89) were prepared by aprocedure similar to that of Example 9.

EXAMPLE 37-89

[0391] TABLE I Example # STRUCTURE MF MW 37

C₄₆H₅₄N₄O₇ 774.954 38

C₅₀H₅₅N₃O₇ 809.998 39

C₅₀H₅₄N₂O₈ 810.983 40

C₄₉H₅₈N₂O₇ 787.004 41

C₅₀H₅₈N₂O₉ 831.013 42

C₄₄H₅₈N₂O₈ 742.948 43

C₄₆H₆₂N₂O₉ 787.001 44

C₄₄H₅₈N₂O₇ 726.949 45

C₄₅H₅₈N₂O₇ 738.96 46

C₄₇H₆₀N₂O₇ 764.998 47

C₄₈H₅₆N₂O₉ 804.975 48

C₄₄H₅₆N₂O₇ 724.933 49

C₄₄H₅₆N₂O₇ 724.933 50

C₅₀H₅₈N₂O₈ 815.014 51

C₄₅H₅₆N₂O₉ 768.942 52

C₄₈H₆₁N₃O₈ 808.023 53

C₄₄H₅₆N₂O₈ 740.932 54

C₄₂H₅₄N₂O₇ 698.896 55

C₄₈H₅₈N₂O₇ 774.993 56

C₄₆H₅₄N₂O₇ 746.94 57

C₄₇H₅₆N₂O₇ 760.966 58

C₄₅H₅₂N₂O₇ 732.913 59

C₄₈H₅₈N₂O₇ 774.993 60

C₅₀H₅₅N₃O₇ 809.998 61

C₄₃H₅₂N₂O₇ 708.891 62

C₄₄H₅₆N₂O₇ 724.933 63

C₄₆H₆₀N₂O₉ 784.985 64

C₄₄H₅₆N₂O₇ 724.933 65

C₄₃H₄₉NO₈ 707.859 66

C₄₇H₅₅NO₈ 761.951 67

C₄₇H₅₅NO₈ 761.951 68

C₄₈H₅₇NO₁₀ 807.975 69

C₄₆H₅₃NO₈ 747.924 70

C₄₆H₅₁NO₉ 761.907 71

C₄₈H₅₇NO₉ 791.976 72

C₄₇H₅₅NO₈ 761.951 73

C₄₆H₆₀N₂O₇ 752.987 74

C₄₅H₅₈N₂O₇ 738.96 75

C₄₇H₅₅NO₉ 777.95 76

C₄₃H₅₃NO₈ 711.891 77

C₄₅H₅₈N₂O₇ 738.96 78

C₄₄H₅₈N₂O₇ 726.949 79

C₄₅H₆₀N₂O₇ 740.976 80

C₄₆H₅₈N₂O₉ 782.969 81

C₄₃H₅₂N₂O₈ 724.89 82

C₄₇H₆₂N₂O₇ 767.014 83

C₅₀H₆₀N₂O₇ 801.031 84

C₄₇H₅₅NO₈ 761.951 85

C₄₃H₅₃NO₈ 711.891 86

C₄₄H₅₇NO₈ 727.933 87

C₄₈H₅₇NO₉ 791.976 88

C₄₇H₅₅NO₉ 777.95 89

C₄₇H₅₄N₂O₉ 790.949

EXAMPLE 90 Identification af Gambogic Acid and Analogs as AntineoplasticCompounds that are Caspase Cascade Activators

[0392] Human breast cancer cell lines T-47D and ZR-75-1, human prostatecancer cell line PC-3, human leukemia cancer cell line HL-60 and humannon-transformed fibroblast cell line MRC-5 cells were grown according tomedia component mixtures designated by The American Type CultureCollection+10% FCS (Life Technologies, Inc.), in a 5% CO₂-95% humidityincubator at 37° C. T-47D, ZR-75-1 and PC-3 cells were maintained at acell density between 30 and 80% confluency and for HL-60 at a celldensity of 0.1 to 0.6×10⁶ cells/m. Cells were harvested at 600× g andresuspended at 0.65×10⁶ cells/ml into appropriate media +10% FCS. Analiquot of 45 μl of cells was added to a well of a 96-well microtiterplate containing 5 μof a 10% DMSO in RPMI-1640 media solution containing1.6 to 100 μM gambogic acid or other test compound (0.16 to 10 μMfinal). An aliquot of 45 μl of cells was added to a well of a 96-wellmicrotiter plate containing 5 μl of a 10% DMSO in RPMI-1640 mediasolution without test compound as the control sample. The samples weremixed by agitation and then incubated at 37° C. for 24 h in a 5% CO₂-95%humidity incubator. After incubation, the samples were removed from theincubator and 50 μl of a solution containing 20 μM ofN-(Ac-DEVD)-N′-ethoxycarbonyl-R110 fluorogenic substrate (SEQ ID NO:1)(Cytovia, Inc.; WO99/18856), 20% sucrose (Sigma), 20 mM DTT (Sigma), 200mM NaCl (Sigma), 40 mM Na PIPES buffer pH 7.2 (Sigma), and 500 μg/mllysolecithin (Calbiochem) was added. The samples were mixed by agitationand incubated at room temperature. Using a fluorescent plate reader(Model 1420 Wallac Instruments), an initial reading (T=0) was madeapproximately 1-2 min after addition of the substrate solution,employing excitation at 485 nm and emission at 530 nm, to determine thebackground fluorescence of the control sample. After the 3 h incubation,the samples were read for fluorescence as above (T=3 h).

[0393] Calculation:

[0394] The Relative Fluorescence Unit values (RFU) were used tocalculate the sample readings as follows:

RFU _((T=3 hr))−Control RFU _((I=0))=Net RFU _((T=3 hr))

[0395] The activity of caspase cascade activation was determined by theratio of the net RFU value for gambogic acid or other test compound tothat of control samples. The EC₅₀ (nM) was determined by a sigmoidaldose-response calculation (Prism 2.0, GraphPad Software Inc.). Thecaspase activity (Ratio) and potency (EC₅₀) are summarized in Table II:TABLE II Caspase Activity and Potency T-47D ZR-75-1 PC-3 HL-60 MRC-5Ratio EC50 Ratio EC50 Ratio EC50 Ratio EC50 Ratio EC50 Example # (nM)(nM) (nM) (nM) (nM) 1 13.6 560 11.9 1400 2.1 1500 6.3 400 17.8 1410 416.8 484 14.9 1640 3.9 1330 7.7 339 27.8 501 5 13.9 210 14.3 783 2.7 9005.2 200 12.6 631 6 12.0 2800 ND ND 4.5 5000 ND ND ND ND 2 16.9 310 14.21160 2.9 1350 5.6 340 22.4 1260 3 7.0 1000 ND ND 2.9 1700 ND ND ND ND 714.4 830 13.4 1650 2.3 1700 ND ND 9.4 1200 9 11.7 990 12.8 2050 3.1 5900ND ND 11.4 1900

[0396] Thus, gambogic acid and its derivatives and analogs areidentified as potent caspase cascade activators and antineoplasticcompounds in this assay.

EXAMPLE 91 Identification of Gambogic Acid and Analogs as AntineoplasticCompounds that Exhibit Inhibition of Cell Proliferation (GI₅₀) and CellDeath (LC₅₀)

[0397] T47D, ZR-75-1, PC-3, human prostate cancer cell line DU-145,human non-small cell lung cancer cell line A-549, human small cell lungcancer cell line SHP-77, HL-60 and MRC-5 cells were grown and harvestedas in Example 90. An aliquot of 90 μl of cells (2.2×10⁴ cells/ml) wasadded to a well of a 96-well microtiter plate containing 10 μl of a 10%DMSO in RPMI-1640 media solution containing 1 nM to 100 μM gambogic acidor other test compound (0.1 nM to 10 μM final). An aliquot of 90 μl ofcells was added to a well of a 96-well microtiter plate containing 10 μlof a 10% DMSO in RPMI-1640 media solution without compound as thecontrol sample for maximal cell proliferation (A_(max)). The sampleswere mixed by agitation and then incubated at 37° C. for 48 h in a 5%CO₂-95% humidity incubator. After incubation, the samples were removedfrom the incubator and 20 μl of CellTiter 96 AQ_(UEOUS) One SolutionCell Proliferation™ reagent (Promega) was added. The samples were mixedby agitation and incubated at 37° C. for 2-4 h in a 5% CO₂-95% humidityincubator. Using an absorbance plate reader (Model 1420 WallacInstruments), an initial reading (T=0) was made approximately 1-2 min.after addition of the solution, employing absorbance at 490 nm. Thisdetermines the possible background absorbance of the test compounds. Noabsorbance for gambogic acid or its analogs or derivatives was found at490 nm. After the 2-4 h incubation, the samples were read for absorbanceas above (A_(Test)).

[0398] Baseline for GI₅₀ (dose for 50% inhibition of cell proliferation)and LC₅₀ (dose for 50% cell death) of initial cell numbers wasdetermined by adding an aliquot of 90 μl of cells or 90 μl of media,respectively, to wells of a 96-well microtiter plate containing 10 μl ofa 10% DMSO in RPMI-1640 media solution. The samples were mixed byagitation and then incubated at 37° C. for 0.5 h in a 5% CO₂-95%humidity incubator. After incubation, the samples were removed from theincubator and 20 μl of CellTiter 96 AQ_(UEOUS) One Solution CellProliferation™ reagent (Promega) was added. The samples were mixed byagitation and incubated at 37° C. for 2-4 h in a 5% CO₂-95% humidityincubator. Absorbance was read as above, (A_(T=0)) defining absorbancefor initial cell number used as baseline in GI₅₀ determinations and(A_(min)) defining absorbance for media alone used as baseline in LC₅₀determinations.

[0399] Calculation:

[0400] GI₅₀ (dose for 50% inhibition of cell proliferation)

50=100×[(A _(Test) −A _(T=0))/(A _(max) −A _(T=0))]

[0401] LC₅₀ (dose for 50% cell death)

50=100×[(A _(Test) −A _(min))/(A _(T=0) −A _(min))]

[0402] The GI₅₀ (nM) and LC₅₀ (nM) are summarized in Table III: TABLEIII GI₅₀ and LC₅₀ in Cancer Cells Methyl-6- Gambogic Methyl GambogylMethoxy- Gambogenic acid Gambogate Piperidine gambogate Acid GambogeninGI₅₀ LC₅₀ GI₅₀ LC₅₀ GI₅₀ LC₅₀ GI₅₀ LC₅₀ GI₅₀ LC₅₀ GI₅₀ LC₅₀ Cell lines(nM) (nM) (nM) (nM) (nM) (nM) T-47D 65 450 40 50 50 50 50 80 500 500 500500 ZR-75-1 400 500 300 500 300 500 400 500 ND ND ND ND PC-3 500 700 500500 500 500 500 500 3000 5000 5000 5000 DU-145 500 800 500 500 500 500500 900 600 5000 2000 5000 A-549 800 5000 500 2000 800 5000 500 900 NDND ND ND SHP-77 500 500 500 500 500 500 500 500 ND ND ND ND HL-60 500700 50 500 100 800 100 800 ND ND ND ND MRC-5 400 500 200 500 800 800 500500 500 5000 3000 5000

[0403] Thus, gambogic acid and its analogs and derivatives areidentified as potent antineoplastic compounds that both inhibit cellproliferation (GI₅₀) and elicit cell death (LC₅₀).

EXAMPLE 92 Identification of Vinblastine, Cisplatin, 5-Fluorouracil,Taxol, Camptothecin, Doxorubicin, Etoposide and Methotrexate asConventional Antineoplastic Agents that are not Efficient CaspaseCascade Activators in Solid Tumors

[0404] T-47D, ZR-75-1, PC-3 and HL-60 cells were grown and harvested asin Example 90. An aliquot of 45 μl of cells was added to a well of a96-well microtiter plate containing 5 μl of a 10% DMSO in RPMI-1640media solution containing 100 μM of test compounds (10 μM final). Analiquot of 45 μl of cells was added to a well of a 96-well microtiterplate containing 5 μl of a 10% DMSO in RPMI-1640 media solution withouttest compound as the control sample. The samples were mixed by agitationand then incubated at 37° C. for 24 h in a 5% CO₂-95% humidityincubator. After incubation, the samples were removed from the incubatorand 50 μl of a solution containing 20 μM ofN-(Ac-DEVD)-N′-ethoxycarbonyl-R110 fluorogenic substrate (SEQ ID NO:1)(Cytovia, Inc.), 20% sucrose (Sigma), 20 mM DTT (Sigma), 200 mM NaCl(Sigma), 40 mM Na PIPES buffer pH 7.2 (Sigma), and 500 μg/mllysolecithin (Calbiochem) was added. The samples were mixed by agitationand incubated for 3 h at room temperature. Using a fluorescent platereader (Model 1420 Wallac Instruments), an initial reading (T=0) wasmade approximately 1-2 min. after addition of the substrate solution,employing excitation at 485 nm and emission at 530 nm, to determine thebackground fluorescence of the control sample. After the 3 h incubation,the samples were read for fluorescence as above (T=3 h).

[0405] Calculation:

[0406] The Relative Fluorescence Unit values (RFU) were used tocalculate the sample readings as follows:

RFU _((T=3 hr))−Control RFU _((T=0))=Net RFU _((T=3 hr))

[0407] The activity in caspase cascade activation was determined by theratio of the net RFU value for test compounds to that of controlsamples. A ratio around 1 indicates that the compound is not anefficient caspase cascade activator. The ratios are summarized in TableIV.

[0408] Table IV. Activity of Known Antineoplastic Compound as CaspaseCascade Activators TABLE IV Activity of Known Antineoplastic Compound asCaspase Cascade Activators Cell lines T-47D PC-3 Vinblastine 0.9 0.8Cisplatin 1.1 0.9 5-fluorouracil 0.8 0.7 Taxol 0.9 0.7 Camptothecin 0.70.6 Doxorubicin 1.3 1.1 Etoposide 1.0 0.8 Methotrexate 0.8 0.7

[0409] Thus, vinblastine, cisplatin, 5-fluorouracil, taxol,camptothecin, doxorubicin, etoposide and methotrexate are identified asknown antineoplastic compounds that are not caspase cascade activatorsin this assay.

EXAMPLE 93 Morphological Change of T47D Cells Treated with Gambogic Acid

[0410] Cells undergoing apoptosis typically demonstrate severalcharacteristic morphological changes, including rounding and blebbing.In addition, apoptotic adherent cells in culture lose their ability toremain attached to the culture dish. The ability of gambogic acid totrigger these morphological changes in T47D cells was investigated.

[0411] 60 mm culture dishes were seeded with 750,000 T47D cells and thecultures were incubated under normal growth conditions (complete mediumwith 10% FBS) for 24 h. The cells were then treated with 2.5 μM ofgambogic acid and further incubated under normal growth conditions for 2or 6 h. Morphological changes were documented by photographing the cellsunder phase contrast illumination.

[0412] As shown in FIGS. 1A-C, T47D cells incubated with vehicle(Control) are phase-dark and show a normal, flat morphology (FIG. 1A).After 2 h of treatment with gambogic acid, many of the cells have takenon a rounded, phase-bright morphology (FIG. 1B). By 6 h of treatmentwith gambogic acid, most of the cells in the culture are rounded up andare beginning to detach from the dish (FIG. 1C). At this timepoint, manyof the cells also show evidence of blebbing. Based on these data, it wasconcluded that gambogic acid induces apoptotic morphological changes inT47D cells.

EXAMPLE 94 Gambogic Acid Induces Nuclear Fragmentation in T47D BreastCancer Cells

[0413] T47D cells were grown and plated as described in Example 90. Thecells were treated with 10 μM of gambogic acid and the plate wasincubated for up to 24 h at 37° C. in a 5% CO₂-95% humidity incubator.At 24 h, the cells were incubated with a live cell nucleic acid stain,Syto16 (Molecular Probes) which stains DNA. After 2 washes with PBS,cells were examined under a fluorescence microscope. The nuclearstaining of untreated cells showed normal nuclei (FIG. 2A) whereas thegambogic acid treated cells showed condensed and fragmented nuclei in alarge population of the cells (FIG. 2B). Nuclear fragmentation is aclear indicator of cellular apoptosis.

EXAMPLE 95 Gambogic Acid Induces Characteristic Apoptotic Morphology inJurkat Cells

[0414] Jurkat T leukemia cells were grown in RPMI 1640 media (LifeTechnologies, Inc.)+10% FCS (Sigma Chemical Company) in a 5% CO₂-95%humidity incubator at 37° C., and maintained at a cell density between 4and 8×10⁵ cells/ml. Cells were harvested at 200× g and resuspended at1-2×10⁶ cells/ml into RPMI 1640 media+10% FCS, and 3 ml of the cells wasdispensed in each of three wells of a 6-well plate. One of the wells wastreated with 10 μM caspase inhibitor cbz-Val-Asp-fink (Cytovia, Inc.;WO99/18781) and the plate was incubated at 37° C. in a 5% CO₂-95%humidity incubator for 1 h prior to addition of gambogic acid. The wellswith and without the caspase inhibitor were treated with 10 μM gambogicacid. The third well was treated with solvent (control cells). The platewas incubated at 37° C. in a 5% CO₂-95% humidity incubator.

[0415] At 30 min. after addition of gambogic acid an aliquot of cellsfrom each well was taken into the capillary slides and observed under aphase-contrast microscope.

[0416] The control cell samples showed normal cell morphology (FIG. 3A)whereas after 30 min. treatment with gambogic acid the cells showedblebbing and cellular fragmentation (FIG. 3B), hallmarks of apoptosis.The presence of caspase inhibitor prevented the morphological changes(FIG. 3C), indicating that the changes are due to activation of caspasesin the cell.

EXAMPLE 96 Activation of Caspases by Gambogic Acid in T47D Breast CancerCell Line and in Normal Fibroblasts MRC-5

[0417] T47D cells and MRC-5 cells were maintained and harvested asdescribed in Example 90. An aliquot of 45 μl of cells was added to eachwell of a 96-well microtiter plate. To determine the dose response ofgambogic acid for inducing caspase activity, 5 μl of 20 μM gambogic acidin RPMI media was added to wells in triplicates. Two-fold serialdilutions were made for the lower concentrations. After incubation for 2h, the samples were removed from the 5% CO₂-95% humidity incubator andcaspase activity was determined by addition of a fluorogenic substrateas described in Example 90.

[0418] The dose response (FIG. 4) indicated that the human breast cancercell line T47D is more sensitive to induction of caspase activity than anormal fibroblast cell line MRC-5 by gambogic acid. Therefore, there isa potential therapeutic index with gambogic acid treatment.

EXAMPLE 97 Gambogic Acid Induces Caspase Activity in a Variety of SolidTumor Cell Lines Which is Inhibited by a Caspase Inhibitor

[0419] T47D, ZR-75, PC3, SHP-77 and A-549 cells were maintained andharvested as described in Example 90. Cells were added to 96-well platesas described in Example 90. The cells were treated with 10 μM gambogicacid, in the presence and in the absence of 10 μM caspase inhibitorcbz-Val-Asp-fmk (Cytovia, Inc.; WO99/18781). The plate was incubated upto 24 h at 37° C in a 5% CO₂-95% humidity incubator. Caspase activitywas determined by addition of a fluorogenic substrate as described inExample 90.

[0420] Gambogic acid induced caspase activity in a ratio of greater than2.5 (+) above untreated cell levels in all the tested cancer cell lines(Table IV). The caspase activity detected was inhibited by the caspaseinhibitor (+), confirming that the fluorescent signal was due to caspaseactivity. TABLE IV Gambogic Acid as Caspase Inducers in Solid TumorCells Caspase Inhibition of caspase Cell line activity activity byInhibitor T47D + + ZR-75 + + PC-3 + + SHP-77 + + A-549 + +

EXAMPLE 98 Induction of PARP Cleavage by Gambogic Acid in Human TumorCells

[0421] Cleavage of the enzyme poly(ADP)ribose polymerase (PARP) bycaspase-3 and related proteases is considered to be one of the molecularhallmarks of caspase-mediated apoptosis. Therefore, the ability ofgambogic acid to induce PARP cleavage in four different human tumor celllines (Jurkat cells, HL-60 cells, T47D cells and PC3 cells) wasdetermined.

[0422] Cells were cultured in complete growth medium containing 10% FBSand treated with gambogic acid at concentrations of 2.5 μM or 5 μM for 2to 4 h. Control cultures were treated with a drug vehicle (DMSO), or thewell-characterized apoptosis inducer, staurosporine. At the end of theapoptosis induction period, the cells were harvested, washed once withPBS, quick-frozen on dry ice, and stored at −80° C. The cells were thenlysed in a standard immunoblotting lysis buffer and samples of thelysates were electrophoresed on 4% to 20% gradient polyacrylamide gels.The proteins in the gels were then transferred to PVDF membranes andprobed with a commercially-available rabbit polyclonal antibody to PARP.

[0423] FIGS. 5A-D illustrate the results of these experiments. A 2 htreatment with 2.5 μM gambogic acid induced almost complete PARPcleavage in both Jurkat cells (FIG. 5A) and HL-60 cells (FIG. 5B). 2.5μM gambogic acid was as effective as 1 μM staurosporine, one of the mostpotent apoptosis inducers known. There was no PARP cleavage in cellstreated with drug vehicle (DMSO) or another inactive control.

[0424]FIG. 5C shows the effect of gambogic acid on PARP cleavage in T47Dcells. Within 2 h of treatment, using 2.5 μM gambogic acid, moderateinduction of PARP cleavage is observed; almost complete PARP cleavage isobserved with 5 μM gambogic acid. Within 4 h of treatment, bothconcentrations of gambogic acid give almost complete PARP cleavage.Under the same conditions, no cleavage of PARP was observed for cellstreated with 1 μM staurosporine.

[0425] PC3 cells, a human prostate cancer cell line, were more resistantto the induction of PARP cleavage by gambogic acid (FIG. 5D). Within 2 hof treatment, neither concentration (2.5 μM and 5 μM) of drug waseffective. Within 4 h of treatment, a moderate amount of PARP cleavageproduct could be observed with the highest dose of gambogic acid (5 μM).Under the same conditions, no cleavage of PARP was observed for cellstreated with 1 μM staurosporine.

[0426] Based on these experiments, it was concluded that gambogic acidtriggers PARP cleavage in all four human tumor cell lines tested. Theseresults indicate that gambogic acid is an effective inducer ofcaspase-mediated apoptosis in tumor cells under normal growthconditions.

[0427] Having now fully described this invention, it will be understoodby those of ordinary skill in the art that the same can be performedwithin a wide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or anyembodiment thereof. All patents, patent applications and publicationscited herein are fully incorporated by reference herein in theirentirety.

1 1 1 4 PRT Artificial Sequence Synthetic peptide 1 Asp Glu Val Asp 1

What is claimed is:
 1. A method of treating a disorder responsive to theinduction of apoptosis in an animal suffering therefrom, comprisingadministering to a mammal in need of such treatment an effective amountof a compound having one of the Formulae I-III:

or a pharmaceutically acceptable salt or prodrug thereof, wherein: thedotted lines are single bonds, double bonds or epoxy groups; X togetherwith the attached carbon is a methylene, carbonyl, hydroxymethinyl,alkoxymethinyl, aminomethinyl, an oxime, a hydrazone, an arylhydrazoneor semicarbazone; Y together with the attached carbon is a methylene,carbonyl, hydroxymethinyl, alkoxymethinyl, aminomethinyl, an oxime, ahydrazone, an arylhydrazone or semicarbazone; R₁ is formyl,methylenehydroxy, carboxy, acyl (R_(a)CO), optionally substitutedalkoxycarbonyl (R_(a)OCO), optionally substituted alkylthiocarbonyl,optionally substituted arninocarbonyl (carbamyl, R_(b)R_(c)NCO) orhydroxyaminocarbonyl, where R_(a) is hydrogen, optionally substitutedlower alkyl, optionally substituted aryl, or optionally substitutedlower aralkyl group; R_(b) and R_(c) are independently hydrogen,optionally substituted heteroalkyl, optionally substituted lower alkyl,optionally substituted aryl, optionally substituted heteroaryl oroptionally substituted lower aralkyl groups; or R_(b) and R_(c) may betaken together with the attached N to form an optionally substituted,saturated or partially saturated 5-7 membered heterocyclo group; R₂ ishydrogen, optionally substituted alkyl, acyl (R_(a)CO), carbamyl(R_(b)R_(c)NCO) or sulfonyl (R_(d)SO₂), where R_(a), R_(b), and R_(c)are defined above; R_(d) is hydrogen, optionally substituted loweralkyl, optionally substituted aryl, or optionally substituted loweraralkyl groups; R₃ is hydrogen or prenyl; R₄ is hydrogen, halogen,hydroxy, optionally substituted alkyl, cycloalkyl, alkoxy, alkylthio oramino; and R₅ is hydrogen, optionally substituted alkyl or acyl(R_(a)CO), carbamyl (R_(b)R_(c)NCO) or sulfonyl (R_(d)SO₂), where R_(a),R_(b), R_(c) and R_(d) are defined above; with the proviso that whensaid disorder is cancer, then said compound is not gambogic acid.
 2. Themethod of claim 1, wherein the dotted lines between C-9 and C-10 of acompound of Formula I or III represent a double bond, R₄ is not acycloalkyl group, the other dotted lines are not epoxy groups, and R_(b)and R_(c) are not heteroalkyl groups.
 3. A compound according to claim1, wherein R₁ is formyl, acetyl, propionyl, carboxy, methoxycarbonyl,ethoxycarbonyl, methylthiocarbonyl, ethylthiocarbonyl,butylthiocarbonyl, dimethylcarbamyl, diethylcarbamyl,N-piperidinylcarbonyl, N-methyl-N′-piperazinylcarbonyl,2(dimethylamino)-ethylcarbamyl or N-morpholinylcarbonyl, and the dottedlines represent double bonds.
 4. A compound according to claim 1,wherein R₂ is hydrogen, formyl, acetyl, dimethylcarbamyl,diethylcarbamyl, 2-(dimethylamino)ethyl-carbamyl, N-piperidinylcarbonyl,N-methyl-N′-piperazinylcarbonyl, N-morpholinylcarbonyl, methylsulfonyl,ethylsulfonyl, phenylsulfonyl, methyl, ethyl, 2-piperidinylethyl,2-morpholinylethyl, 2-(dimethylamino)ethyl, or 2-(diethylamino)ethyl,and the dotted lines represent double bonds.
 5. A compound according toclaim 1, wherein R₄ is methyl, ethyl, phenyl, chloro, bromo, hydroxy,hydrogen, methoxy, ethoxy, methylthio, ethylthio, butylthio,dimethylamino, diethylamino, piperidinyl, pyrrolidinyl, imidazolyl,pyrazolyl, N-methylpiperazinyl, 2-(dimethylamino)ethylamino ormorpholinyl, and the dotted lines represent double bonds.
 6. A compoundaccording to claim 1, wherein R₅ is hydrogen, acetyl, dimethylcarbamyl,diethylcarbamyl, 2-(dimethylamino)ethylcarbamyl, N-piperidinylcarbonyl,N-methyl-N′-piperazinylcarbonyl, N-morpholinyl-carbonyl, methylsulfonyl,ethylsulfonyl, phenylsulfonyl, methyl, ethyl, 2-piperidinylethyl,2-morpholinylethyl, 2-(dimethylamino)ethyl, or 2-(diethylamino)ethyl. 7.The method according to claim 1, wherein said compound is selected fromthe group consisting of: Gambogic acid; Methyl gambogate;9,10-Dihydrogambogic acid; 9,10-Dihydrogambogyl (4-methylpiperazine);9,10-Dihydrogambogyl (2-dimethylaminoethylamine);9,10-Dihydro-12-hydroxygambogic acid; Gambogyl diethylamine; Gambogyldimethylamine; Gambogyl amine; Gambogyl hydroxyamine; Gambogylpiperidine; 6-Methoxy-gambogic acid; 6-(2-Dimethylaminoethoxy)-gambogicacid; 6-(2-Piperidinylethoxy)-gambogic acid;6-(2-Morpholinylethoxy)-gambogic acid; 6-Methoxy-gambogyl piperidine;Gambogyl morpholine; Gambogyl (2-dimethylaminoethylamine);10-Morpholinyl-gambogyl morpholine; 10-Morpholinyl-gambogyl piperidine;10-(4-Methylpiperazinyl)-gambogyl piperidine;10-(4-Methylpiperazinyl)-gambogyl morpholine; 10-Piperidinyl-gambogylpiperidine; 10-(4-Methylpiperazinyl)-gambogyl (4-methylpiperazine);10-Cyclohexyl gambogic acid; 10-Methyl gambogic acid; Gambogyl(4-methylpiperazine); Methyl-6-Methoxy-gambogate; Gambogenic acid;Gambogenin; 10-Methoxy-gambogic acid; 10-Butylthio-gambogic acid;10-(4-Methylpiperazinyl)-gambogic acid; 10-Pyrrolidinyl-gambogic acid;Methyl-10-Morpholinyl-gambogate; 10-Piperidinyl-gambogic acid;10-Morpholinyl-gambogic acid; N-(2-Gambogylamido-ethyl)biotinamide;Gambogyl (2-(4-morpholinyl)ethylamine); 9,10-Epoxygambogic acid;Gambogyl (4-(2-pyridyl)piperazine);10-(4-(2-Pyridyl)piperazinyl)gambogyl (4-(2-pyridyl)piperazine);6-Acetylgambogic acid; 10-(4-(2-Pyridyl)piperazinyl)gambogic acid;8-(Gambogylamido)octanoic acid; 6-(Gambogylamido)hexanoic acid;12-(Gambogylamido)dodecanoic acid; 10-Methoxy-gambogyl piperidine;Gambogyl (4-(2-pyrimidyl)piperazine); Gambogyl(bis(2-pyridylmethyl)amine); Gambogyl(N-(3-pyridyl)-N-(2-hydroxybenzyl)amine); Gambogyl (4-benzylpiperazine);Gambogyl (4-(3,4-methylenedioxybenzyl)piperazine); Gambogyl(N-methyl-5-(methylamino)-3-oxapentylamine); Gambogyl(N-methyl-8-(methylamino)-3,6-dioxaoctylamine); Gambogyl(N-ethyl-2-(ethylamino)ethylamine); Gambogyl (4-isopropylpiperazine);Gambogyl (4-cyclopentylpiperazine); Gambogyl(N-(2-oxo-2-ethoxyethyl)-(2-pyridyl)methylamine); Gambogyl(2,5-dimethylpiperazine); Gambogyl (3,5-dimethylpiperazine); Gambogyl(4-(4-acetylphenyl)piperazine); Gambogyl (4-ethoxycarbonylpiperazine);Gambogyl (4-(2-oxo-2-pyrrolidylethyl)piperazine); Gambogyl(4-(2-hydroxyethyl)piperazine); Gambogyl(N-methyl-2-(methylamino)ethylamine); Gambogyl(N-methyl-2-(benzylamino)ethylamine); Gambogyl(N-methyl-(6-methyl-2-pyridyl)methylamine); Gambogyl(N-ethyl-2-(2-pyridyl)ethylamine); Gambogyl(N-methyl-(2-pyridyl)methylamine); Gambogyl(N-methyl-4-(3-pyridyl)butylamine); Gambogyl(bis(3-pyridylmethyl)amine); Gambogyl (2,4-dimethyl-2-imidazoline);Gambogyl (4-methyl-homopiperazine); Gambogyl(4-(5-hydroxy-3-oxapentyl)piperazine); Gambogyl(3-dimethylaminopyrrolidine); Gambogyl ((2-furanyl)methylamine);Gambogyl (2-hydroxy-1-methyl-2-phenylethylamine); Gambogyl(3,4,5-trimethoxybenzylamine); Gambogyl (2-(2-methoxyphenyl)ethylamine);Gambogyl (2-methoxybenzylamine); Gambogyl(3,4-methylenedioxybenzylamine); Gambogyl(2-(2,5-dimethoxyphenyl)ethylamine); Gambogyl(2-(3-methoxyphenyl)ethylamine); Gambogyl (3-(piperidinyl)propylamine);Gambogyl (2-(piperidinyl)ethylamine); Gambogyl(3,4-dimethoxybenzylamine); Gambogyl ((2-tetrahydrofuranyl)methylamine);Gambogyl ((N-ethyl-2-pyrrolidinyl)methylamine); Gambogyl(2-diethylaminoethylamine); Gambogyl(2,2-dimethyl-3-(dimethylaminopropylamine); Gambogyl((N-ethoxycarbonyl-4-piperidinyl)amine); Gambogyl(2-carbamylpyrrolidine); Gambogyl (3-(homopiperidinyl)propylamine);Gambogyl ((N-benzyl-4-piperidinyl)amine); Gambogyl(2-(4-methoxyphenyl)ethylamine); Gambogyl (4-oxa-hex-5-enylamine);Gambogyl (6-hydroxyhexylamine); Gambogyl(2-(3,5-dimethoxyphenyl)ethylamine); Gambogyl(3,5-dimethoxybenzylamine); and Gambogyl(2-carbamyl-2-(4-hydroxyphenyl)ethylamine).
 8. A method for treating orpreventing cancer, comprising administering to an animal in need of suchtreatment an effective amount of a compound having one of the FormulaeI-III:

or a pharmaceutically acceptable salt or prodrug thereof, wherein: thedotted lines are single bonds, double bonds or epoxy groups; X togetherwith the attached carbon is a methylene, carbonyl, hydroxymethinyl,alkoxymethinyl, aminomethinyl, an oxime, a hydrazone, an arylhydrazoneor semicarbazone; Y together with the attached carbon is a methylene,carbonyl, hydroxymethinyl, alkoxymethinyl, aminomethinyl, an oxime, ahydrazone, an arylhydrazone or semicarbazone; R₁ is formyl,methylenehydroxy, carboxy, acyl (R_(a)CO), optionally substitutedalkoxycarbonyl (R_(a)OCO), optionally substituted alkylthiocarbonyl,optionally substituted aminocarbonyl (carbamyl, R_(b)R_(c)NCO) orhydroxyaminocarbonyl, where R_(a) is hydrogen, optionally substitutedlower alkyl, optionally substituted aryl, or optionally substitutedlower aralkyl group; R_(s) and R_(c) are independently hydrogen,optionally substituted heteroalkyl, optionally substituted lower alkyl,optionally substituted aryl, optionally substituted heteroaryl oroptionally substituted lower aralkyl groups; or R_(b) and R_(c) may betaken together with the attached N to form an optionally substituted,saturated or partially saturated 5-7 membered heterocyclo group; R₂ ishydrogen, optionally substituted alkyl, acyl (R_(a)CO), carbamyl(R_(b)R_(c)NCO) or sulfonyl (R_(d)SO₂), where R_(a), R_(b) and R_(c) aredefined above; R_(s) is hydrogen, optionally substituted lower alkyl,optionally substituted aryl, or optionally substituted lower aralkylgroups; R₃ is hydrogen or prenyl; R₄ is hydrogen, halogen, hydroxy,optionally substituted alkyl, cycloalkyl, alkoxy, alkylthio or amino;and R₅ is hydrogen, optionally substituted alkyl or acyl (R_(a)CO),carbamyl (R_(b)R_(c)NCO) or sulfonyl (R_(d)SO2), where R_(a), R_(b),R_(c) and R_(d) are defined above; with the proviso that said compoundis not gambogic acid.
 9. The method of claim 8, wherein the dotted linesbetween C-9 and C-10 of a compound of Formula I or III represent adouble bond, R₄ is not a cycloalkyl group, the other dotted lines arenot epoxy groups, and R_(b) and R^(c) are not heteroalkyl groups. 10.The method according to claim 8, wherein the method is for treating orpreventing Hodgkin's disease, non-Hodgkin's lymphomas, acute and chroniclymphocytic leukemias, multiple myeloma, neuroblastoma, breastcarcinomas, ovarian carcinomas, lung carcinomas, Wilms' tumor, cervicalcarcinomas, testicular carcinomas, soft-tissue sarcomas, chroniclymphocytic leukemia, primary macroglobulinemia, bladder carcinomas,chronic granulocytic leukemia, primary brain carcinomas, malignantmelanoma, small-cell lung carcinomas, stomach carcinomas, coloncarcinomas, malignant pancreatic insulinoma, malignant carcinoidcarcinomas, malignant melanomas, choriocarcinomas, mycosis fungoides,head and neck carcinomas, osteogenic sarcoma, pancreatic carcinomas,acute granulocytic leukemia, hairy cell leukemia, neuroblastoma,rhabdomyosarcoma, Kaposi's sarcoma, genitourinary carcinomas, thyroidcarcinomas, esophageal carcinomas, malignant hypercalcemia, cervicalhyperplasia, renal cell carcinomas, endometrial carcinomas, polycythemiavera, essential thrombocytosis, adrenal cortex carcinomas, skin cancer,or prostatic carcinomas.
 11. A method for the treatment of drugresistant cancer, comprising administering to an animal in need of suchtreatment an effective amount of a compound having one of the FormulaeI-III:

or a pharmaceutically acceptable salt or prodrug thereof, wherein: thedotted lines are single bonds, double bonds or epoxy groups; X togetherwith the attached carbon is a methylene, carbonyl, hydroxymethinyl,alkoxymethinyl, aminomethinyl, an oxime, a hydrazone, an arylhydrazoneor semicarbazone; Y together with the attached carbon is a methylene,carbonyl, hydroxymethinyl, alkoxymethinyl, aminomethinyl, an oxime, ahydrazone, an arylhydrazone or semicarbazone; R₁ is formyl,methylenehydroxy, carboxy, acyl (R_(a)CO), optionally substitutedalkoxycarbonyl (R_(a)OCO), optionally substituted alkylthiocarbonyl,optionally substituted aminocarbonyl (carbamyl, R_(b)R_(c)NCO) orhydroxyaminocarbonyl, where R_(a) is hydrogen, optionally substitutedlower alkyl, optionally substituted aryl, or optionally substitutedlower aralkyl group; R_(b) and R_(c) are independently hydrogen,optionally substituted heteroalkyl, optionally substituted lower alkyl,optionally substituted aryl, optionally substituted heteroaryl oroptionally substituted lower aralkyl groups; or R_(b) and R_(c) may betaken together with the attached N to form an optionally substituted,saturated or partially saturated 5-7 membered heterocyclo group; R₂ ishydrogen, optionally substituted alkyl, acyl (R_(a)CO), carbamyl(R_(b)R_(c)NCO) or sulfonyl (R_(d)SO₂), where R_(a), R_(b) and R_(c) aredefined above; R_(d) is hydrogen, optionally substituted lower alkyl,optionally substituted aryl, or optionally substituted lower aralkylgroups; R₃ is hydrogen or prenyl; R₄ is hydrogen, halogen, hydroxy,optionally substituted alkyl, cycloalkyl, alkoxy, alkylthio or amino;and R₅ is hydrogen, optionally substituted alkyl or acyl (R_(a)CO),carbamyl (R_(b)R_(c)NCO) or sulfonyl (R_(d)SO₂), where R_(a), R_(b),R_(c) and R_(d) are defined above.
 12. The method of claim 11, whereinthe dotted lines between C-9 and C-10 of a compound of Formula I or IIIrepresent a double bond, R₄ is not a cycloalkyl group, the other dottedlines are not epoxy groups, and R_(b) and R_(c) are not heteroalkylgroups.
 13. The method according to claim 8 or 11, wherein said compoundis administered together with at least one known cancer chemotherapeuticagent, or a pharmaceutically acceptable salt of said agent.
 14. Themethod according to claim 13, wherein said known cancer chemotherapeuticagent is selected from the group consisting of busulfan, cisplatin,mitomycin C, carboplatin, colchicine, vinblastine, paclitaxel,docetaxel, camptothecin, topotecan, doxorubicin, etoposide,5-azacytidine, 5-fluorouracil, methotrexate, 5-fluoro-2′-deoxy-uridine,ara-C, hydroxyurea, thioguanine, melphalan, chlorambucil,cyclophosamide, ifosfamide, vincristine, mitoguazone, epirubicin,aclarubicin, bleomycin, mitoxantrone, elliptinium, fludarabine,octreotide, retinoic acid, tamoxifen, Herceptin, Rituxan and alanosine.15. The method according to claim 8 or 11, wherein said animal is alsotreated with radiation-therapy.
 16. The method according to claim 8 or11, wherein said compound(s) are administered after surgical treatmentfor cancer.
 17. The method according to claim 1, wherein said disorderis an autoimmune disease.
 18. The method according to claim 1, whereinsaid disorder is rheumatoid arthritis.
 19. The method according to claim1, wherein said disorder is inflammation or inflammatory bowel disease.20. The method according to claim 1, wherein said disorder is a skindisease.
 21. The method according to claim 20, wherein said skin diseaseis psoriasis.
 22. A compound having the Formula I:

or a pharmaceutically acceptable salt or prodrug thereof, wherein: thedotted lines are single bonds, double bonds or epoxy groups; X togetherwith the attached carbon is a methylene, carbonyl, hydroxymethinyl,alkoxymethinyl, aminomethinyl, an oxime, a hydrazone, an arylhydrazoneor semicarbazone; Y together with the attached carbon is a methylene,carbonyl, hydroxymethinyl, alkoxymethinyl, aminomethinyl, an oxime, ahydrazone, an arylhydrazone or semicarbazone; R₁ is formyl,methylenehydroxy, carboxy, acyl (R_(a)CO), optionally substitutedalkoxycarbonyl (R_(a)OCO), optionally substituted alkylthiocarbonyl,optionally substituted aminocarbonyl (carbamyl, R_(b)R_(c)NCO) orhydroxyaminocarbonyl, where R_(a) is hydrogen, optionally substitutedlower alkyl, optionally substituted aryl, N-succinimidyl or optionallysubstituted lower aralkyl group; R_(b) and R_(c) are independentlyhydrogen, optionally substituted heteroalkyl, optionally substitutedlower alkyl, optionally substituted aryl, optionally substitutedheteroaryl or optionally substituted lower aralkyl groups; or R_(b) andR_(c) may be taken together with the attached N to form an optionallysubstituted, saturated or partially saturated 5-7 membered heterocyclogroup; R₂ is hydrogen, optionally substituted alkyl, acyl (R_(a)CO),carbamyl (R_(b)R_(c)NCO) or sulfonyl (R_(d)SO₂), where R_(a), R_(b) andR_(c) are defined above; R_(d) is hydrogen, optionally substituted loweralkyl, optionally substituted aryl, or optionally substituted loweraralkyl groups; and R₃ is hydrogen or prenyl; with the proviso that ifR₁ is carboxy or methoxycarbonyl, X and Y are O, and R₃ is prenyl, thenR₂ is not a hydrogen or methyl.
 23. A compound of claim 22, wherein thedotted lines between C-9 and C-10 represent a double bond, the otherdotted lines are not epoxy groups, and R_(b) and R_(c) are notheteroalkyl groups.
 24. A compound according to claim 22, wherein R₁ isformyl, acetyl, propionyl, carboxy, methoxycarbonyl, ethoxycarbonyl,methylthiocarbonyl, ethylthiocarbonyl, butylthiocarbonyl,dimethylcarbamyl, diethylcarbamyl, N-piperidinylcarbonyl,N-methyl-N′-piperazinylcarbonyl, 2-(dimethylamino)-ethylcarbamyl orN-morpholinylcarbonyl, and the dotted lines represent double bonds. 25.A compound according to claim 22, wherein R₂ is hydrogen, formyl,acetyl, dimethylcarbamyl, diethylcarbamyl,2-(dimethylamino)ethylcarbamyl, N-piperidinylcarbonyl,N-methyl-N′-piperazinylcarbonyl, N-morpholinylcarbonyl, methylsulfonyl,ethylsulfonyl, phenylsulfonyl, methyl, ethyl, 2-piperidinylethyl,2-morpholinylethyl, 2-(dimethylamino)ethyl, or 2-(diethylamino)ethyl,and the dotted lines represent double bonds.
 26. A compound having theFormula II:

or a pharmaceutically acceptable salt or prodrug thereof, wherein: thedotted lines are single bonds, double bonds or epoxy groups; X togetherwith the attached carbon is a methylene, carbonyl, hydroxymethinyl,alkoxymethinyl, aminomethinyl, an oxime, a hydrazone, an arylhydrazoneor semicarbazone; Y together with the attached carbon is a methylene,carbonyl, hydroxymethinyl, alkoxymethinyl, aminomethinyl, an oxime, ahydrazone, an arylhydrazone or semicarbazone; R₁ is formyl,methylenehydroxy, carboxy, acyl (R_(a)CO), optionally substitutedalkoxycarbonyl (R_(a)OCO), optionally substituted alkylthiocarbonyl,optionally substituted aminocarbonyl (carbamyl, R_(b)R_(c)NCO) orhydroxyaminocarbonyl, where R_(a) is hydrogen, optionally substitutedlower alkyl, optionally substituted aryl, N-succinimidyl or optionallysubstituted lower aralkyl group; R_(b) and R_(c) are independentlyhydrogen, optionally substituted heteroalkyl, optionally substitutedlower alkyl, optionally substituted aryl, optionally substitutedheteroaryl or optionally substituted lower aralkyl groups; or R_(b) andR_(c) may be taken together with the attached N to form an optionallysubstituted, saturated or partially saturated 5-7 membered heterocyclogroup; R₂ is hydrogen, optionally substituted alkyl, acyl (R_(a)CO),carbamyl (R_(b)R_(c)NCO) or sulfonyl (R_(d)SO₂), where R_(a), R_(b) andR_(c) are defined above; R_(d) is hydrogen, optionally substituted loweralkyl, optionally substituted aryl, or optionally substituted loweraralkyl groups; R₃ is hydrogen or prenyl; and R₄ is hydrogen, halogen,hydroxy, optionally substituted alkyl, cycloalkyl, alkoxy, alkylthio oramino; with the proviso that if R₁ is formyl or carboxy, R₂ is hydrogen,R₃ is hydrogen, and X and Y are O, then R₄ is not a methoxy or ethoxy.27. A compound of claim 26, wherein R₄ is not a cycloalkyl group, thedotted lines are not epoxy groups, and R_(b) and R_(c) are notheteroalkyl groups.
 28. A compound according to claim 26, wherein R₁ isformyl, acetyl, propionyl, carboxy, methoxycarbonyl, ethoxycarbonyl,methylthiocarbonyl, ethylthiocarbonyl, butylthiocarbonyl,dimethylcarbamyl, diethylcarbamyl, N-piperidinylcarbonyl,N-methyl-N′-piperazinylcarbonyl, 2-(dimethylamino)-ethylcarbamyl orN-morpholinylcarbonyl, and the dotted lines represent double bonds. 29.A compound according to claim 26, wherein R₂ is hydrogen, formyl,acetyl, dimethylcarbamyl, diethylcarbamyl,2-(dimethylamino)ethylcarbamyl, N-piperidinylcarbonyl,N-methyl-N′-piperazinylcarbonyl, N-morpholinylcarbonyl, methylsulfonyl,ethylsulfonyl, phenylsulfonyl, methyl, ethyl, 2-piperidinylethyl,2-morpholinylethyl, 2-(dimethylamino)ethyl, or 2-(diethylamino)ethyl,and the dotted lines represent double bonds.
 30. A compound according toclaim 26, wherein R₄ is methyl, ethyl, phenyl, chloro, bromo, hydroxy,hydrogen, methoxy, ethoxy, methylthio, ethylthio, butylthio,dimethylamino, diethylamino, piperidinyl, pyrrolidinyl, imidazolyl,pyrazolyl, N-methylpiperazinyl, 2(dimethylamino)ethylamino ormorpholinyl, and the dotted lines represent double bonds.
 31. A compoundaccording to claim 22, wherein said compound is selected from the groupconsisting of: Gambogyl dimethylamine; Gambogyl amine; Gambogyldiethylamine; Gambogyl hydroxyamine; Gambogyl piperidine;6-Methoxy-gambogic acid; 6-(2-Dimethylaminoethoxy)-gambogic acid;6-(2-Piperidinylethoxy)-gambogic acid; 6-(2-Morpholinylethoxy)-gambogicacid; 6-Methoxy-gambogyl piperidine; Gambogyl morpholine; Gambogyl(2-dimethylaminoethylamine); Gambogyl (4-methylpiperazine);N-(2-Gambogylamido-ethyl)biotinamide; Gambogyl(2-(4-morpholinyl)ethylamine); Gambogyl (4-(2-pyridyl)piperazine);6-Acetylgambogic acid; N-Hydroxysuccinimidyl gambogate;8-(Gambogylamido)octanoic acid; 6-(Gambogylamido)hexanoic acid;12-(Gambogylamido)dodecanoic acid;N-Hydroxysuccinimidyl-8-(gambogylamido)octanoate;N-Hydroxysuccinimidyl-6-(gambogylamido)hexanoate;N-Hydroxysuccinimidyl-12-(gambogylamido)dodecanoate; Gambogyl(4-(2-pyrimidyl)piperazine); Gambogyl (bis(2-pyridylmethyl)amine);Gambogyl (N-(3-pyridyl)-N-(2-hydroxybenzyl)amine); Gambogyl(4-benzylpiperazine); Gambogyl (4-(3,4-methylenedioxybenzyl)piperazine);Gambogyl (N-methyl-5-(methylamino)-3-oxapentylamine); Gambogyl(N-methyl-8-(methylamino)-3,6-dioxaoctylamine); Gambogyl(N-ethyl-2-(ethylamino)ethylamine); Gambogyl (4-isopropylpiperazine);Gambogyl (4-cyclopentylpiperazine); Gambogyl(N-(2-oxo-2-ethoxyethyl)-(2-pyridyl)methylamine); Gambogyl(2,5-dimethylpiperazine); Gambogyl (3,5-dimethylpiperazine); Gambogyl(4-(4-acetylphenyl)piperazine); Gambogyl (4-ethoxycarbonylpiperazine);Gambogyl (4-(2-oxo-2-pyrrolidylethyl)piperazine); Gambogyl(4-(2-hydroxyethyl)piperazine); Gambogyl(N-methyl-2-(methylamino)ethylamine); Gambogyl(N-methyl-2-(benzylamino)ethylamine); Gambogyl(N-methyl-(6-methyl-2-pyridyl)methylamine); Gambogyl(N-ethyl-2-(2-pyridyl)ethylamine); Gambogyl(N-methyl-(2-pyridyl)methylamine); Gambogyl(N-methyl-4-(3-pyridyl)butylamine); Gambogyl(bis(3-pyridylmethyl)amine); Gambogyl (2,4-dimethyl-2-imidazoline);Gambogyl (4-methyl-homopiperazine); Gambogyl(4-(5-hydroxy-3-oxapentyl)piperazine); Gambogyl(3-dimethylaminopyrrolidine); Gambogyl ((2-furanyl)methylamine);Gambogyl (2-hydroxy-1-methyl-2-phenylethylamine); Gambogyl(3,4,5-trimethoxybenzylamine); Gambogyl (2-(2-methoxyphenyl)ethylamine);Gambogyl (2-methoxybenzylamine); Gambogyl(3,4-methylenedioxybenzylamine); Gambogyl(2-(2,5-dimethoxyphenyl)ethylamine); Gambogyl(2-(3-methoxyphenyl)ethylamine); Gambogyl (3-(piperidinyl)propylamine);Gambogyl (2-(piperidinyl)ethylamine); Gambogyl(3,4-dimethoxybenzylamine); Gambogyl ((2-tetrahydrofuranyl)methylamine);Gambogyl ((N-ethyl-2-pyrrolidinyl)methylamine); Gambogyl(2-diethylaminoethylamine); Gambogyl(2,2-dimethyl-3-dimethylaminopropylamine); Gambogyl((N-ethoxycarbonyl-4-piperidinyl)amine); Gambogyl(2-carbamylpyrrolidine); Gambogyl (3-(homopiperidinyl)propylamine);Gambogyl ((N-benzyl-4-piperidinyl)amine); Gambogyl(2-(4-methoxyphenyl)ethylamine); Gambogyl (4-oxa-hex-5-enylamine);Gambogyl (6-hydroxyhexylamine); Gambogyl(2-(3,5-dimethoxyphenyl)ethylamine); Gambogyl(3,5-dimethoxybenzylamine); and Gambogyl(2-carbamyl-2-(4-hydroxyphenyl)ethylamine).
 32. A compound according toclaim 26, wherein said compound is selected from the group consistingof: 9,10-Dihydrogambogic acid; 9,10-Dihydrogambogyl(4-methylpiperazine); 9,10-Dihydrogambogyl (dimethylamino)ethylamine;9,10-Dihydro-12-hydroxygambogic acid; 10-(4-Methylpiperazinyl)-gambogylpiperidine; 10-(4-Methylpiperazinyl)-gambogyl morpholine;10-Piperidinyl-gambogyl piperidine; 10-(4-Methylpiperazinyl)-gambogyl(4-methylpiperazine); 10-(4-Methylpiperazinyl)-gambogic acid;10-Pyrrolidinyl-gambogic acid; Methyl-10-Morpholinyl-gambogate;10-Morpholinyl-gambogyl morpholine; 10-Morpholinyl-gambogyl piperidine;10-Methoxy-gambogic acid; 10-Butylthio-gambogic acid;10-Piperidinyl-gambogic acid; 10-Morpholinyl-gambogic acid;10-Cyclohexyl-gambogic acid; 10-Methyl-gambogic acid;10-Methoxy-gambogyl piperidine; 10-(4-(2-Pyridyl)piperazinyl)gambogyl(4-(2-pyridyl)piperazine); 10-(4-(2-Pyridyl)piperazinyl)gambogic acid;and 9,10-Epoxygambogic acid.
 33. A pharmaceutical composition,comprising a compound of claim 22 or 26, and a pharmaceuticallyacceptable carrier.
 34. The pharmaceutical composition of claim 33,further comprising at least one known cancer chemotherapeutic agent, ora pharmaceutically acceptable salt of said agent.
 35. The pharmaceuticalcomposition of claim 33, wherein said known cancer chemotherapeuticagentis selected from the group consisting of busulfan, cis-platin,mitomycin C, carboplatin, colchicine, vinblastine, paclitaxel,docetaxel, camptothecin, topotecan, doxorubicin, etoposide,5-azacytidine, 5-fluorouracil, methotrexate, 5-fluoro-2′-deoxy-uridine,ara-C, hydroxyurea, thioguanine, melphalan, chlorambucil,cyclophosamide, ifosfamide, vincristine, mitoguazone, epirubicin,aclarubicin, bleomycin, -mitoxantrone, elliptinium, fludarabine,octreotide, retinoic acid, tamoxifen, Herceptin, Rituxan and alanosine.